Methods for screening agents that modulate presenilin activity and A-β production

ABSTRACT

The disclosure relates generally to neurodegenerative disorders and more specifically to a group of presenilin/G-protein/c-src binding polypeptides and methods of use for modulating signaling and progression of Alzheimer&#39;s disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/788,524 filed Mar. 31, 2006, the disclosure of which isincorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH

The U.S. Government may have certain rights in this invention pursuantto Grant No. AG07888, NS027580, and NS044768 by the National Institutesof Health.

FIELD OF THE INVENTION

The invention relates generally to treating neurodegenerative disordersand more specifically to a group of presenilin/G-protein/c-src bindingpolypeptides and small molecule drugs designed to modulate thephysiologic interactions of polypeptides required for the production ofβ-amyloid (Aβ).

BACKGROUND

The presenilin (PS) proteins are ubiquitous polytopic integral membraneproteins that among other functions, are involved in the development ofneurodegenerative disorders such as Alzheimer's disease (AD) and Down'ssyndrome (DS). AD is a degenerative disorder of the human centralnervous system characterized by progressive memory impairment andcognitive and intellectual decline during mid to late adult life. Thedisease is accompanied by a variety of neuropathologic featuresprincipal among which are the presence in the brain of amyloid plaquesand the neurofibrillary degeneration of neurons. The etiology of thisdisease is complex, although in about 10% of AD cases it appears to befamilial, being inherited as an autosomal dominant trait. Among theseinherited forms of AD, there are at least four different genes, some ofwhose mutants confer inherited susceptibility to this disease. The σ4(Cys112Arg) allelic polymorphism of the Apolipoprotein E (ApoE) gene hasbeen associated with AD in a significant proportion of cases with onsetlate in life. A very small proportion of familial cases with onsetbefore age 65 years have been associated with mutations in the β-amyloidprecursor protein (APP) gene on chromosome 21. A third locus associatedwith a larger proportion of cases with early onset AD has recently beenmapped to chromosome 14q24.3. The majority (70-80%) of heritable,early-onset AD maps to chromosome 14 and appears to result from one ofmore than 20 different amino-acid substitutions within the proteinpresenilin-1 (PS1). A similar, although less common, AD-risk locus onchromosome 1 encodes a protein, presenilin-2 (PS-2, highly homologous toPS-1). Based upon mRNA detection, the presenilins appear to beubiquitously expressed proteins, suggesting that they are normallyhousekeeping proteins required by many cell types.

Presenilin 1 is a 43-45 kDa polypeptide and presenilin 2 is a 53-55 kDapolypeptide. Presenilins are integral proteins of membranes present inhigh molecular weight complexes that are detergent sensitive. Threeprotein components of the complexes in addition to presenilin are known.

The functions of these interacting proteins could influence the specificintercellular binding of β-APP with PS, but so far no familialAlzheimer's disease (FAD) cases have been found where any of these threeproteins are mutated. Missense mutations of presenilin 1 appear todestabilize and cause defective intracellular trafficking of β-catenin.Thus, differential interactions between presenilin polypeptides andproteins capable of specifically binding to presenilins may controlparticular roles of the normal and mutant forms of the presenilinpolypeptides during development.

SUMMARY OF THE INVENTION

This invention provides methods and compositions for identifying agentsthat modulate activity of presenilins. Accordingly, the methods andcompositions provided herein may be used to modulate the production ofAβ in the brain by (1): interfering with the binding of theextra-cellular N-terminal domain of β-APP with PS-1 or PS-2; or (2) byusing as an inhibiting agent a small peptidomimetic molecule, or a smallfragment of an antibody molecule directed to an epitope on either theinteracting surfaces of the β-APP or PS molecules. In one aspect, thepeptide is a soluble N-terminal domain of PS-1 or -2.

In one embodiment, a method of identifying an agent that modulatespresenilin G-protein coupled receptor (GPCR) activity is provided. Themethod includes a) contacting presenilin, or fragment thereof, with aG-protein under conditions that would permit binding of the G-protein topresenilin; b) prior to, simultaneously with, or subsequent to a),contacting presenilin, or fragment thereof, with an agent; c) monitoringpresenilin-mediated binding to the G-protein; and d) determining whetherthe agent modulates presenilin binding to the G-protein therebyidentifying an agent that modulates presenilin G-protein coupledreceptor (GPCR) activity. In some aspects the modulating is byinhibition of presenilin binding to the G-protein. In other aspects, themodulating is by activating presenilin binding to the G-protein. Thepresenilin can be presenilin-1 (PS-1) or presenilin-2 (PS-2). TheG-protein can be G_(o), G_(s), G_(i), G_(z) or G_(q).

In some aspects, the agent includes a naturally occurring or syntheticpolypeptide or oligopeptide, a peptidomimetic, a small organic molecule,a polysaccharide, a lipid, a fatty acid, a polynucleotide, an RNAi orsiRNA, an asRNA, or an oligonucleotide.

The methods provided herein may be conducted in vitro or in vivo. Insome aspects, a method further includes contacting the presenilin withβ-APP prior to, simultaneously with, or subsequent to contacting thepresenilin with the G-protein.

In another embodiment, a method of identifying an agent that modulatespresenilin-mediated Src protein kinase activity is provided. The methodincludes a) contacting presenilin, or fragment thereof, with β-APP underconditions that would permit binding of β-APP to presenilin; b) priorto, simultaneously with, or subsequent to a), contacting presenilin, orfragment thereof, with an agent; c) monitoring presenilin-mediated Srcprotein kinase activity; and d) determining whether the agent modulatespresenilin-mediated Src protein kinase activity.

Also provided herein are compositions and methods for treatingneurodegenerative disorders, and more specifically to a group ofpresenilin/G-protein/c-src binding polypeptides and small molecule drugsdesigned to modulate the physiologic interactions of polypeptidesrequired for the production of β-amyloid (Aβ). the oligopeptide that isthe primary neurotoxic agent in Alzheimer's disease (AD). The objectiveis to reduce the amount of Aβ in the brain to an extent thatsignificantly decreases the neurotoxicity in AD, or delays the onset, ordecreases the severity of the disease. and methods of use for modulatingsignaling and progression of Alzheimer's Disease and improve memory

The invention also provides a method of inhibiting the production of Aβwith a small molecule agent that inhibits the interaction of PS-1 orPS-2 with the G-proteins G_(oA) and G_(oB). The cytoplasmic C-terminaland other domains of PS-1 or PS-2 have been shown by us to be the sitesof interaction of G_(oA) and/or G_(oB) with PS, and that this G_(o)-PSintracellular binding is required for subsequent Aβ production,presumably via the downstream results of this binding process.

The invention similarly provides a method of inhibiting the productionof Aβ by contacting a cell expressing a PS-1 and/or PS-2 with an agentthat interferes with the downstream results of PS-1 and/or PS-2 bindingto G_(o) such as G_(o) activation with phospholipase C.

The invention also provides a method of inhibiting the production of Aβby the use of small molecules, peptides or antibodies selected tointerfere with the activities of members of the Src family of tyrosinekinases.

The invention further provides a method of assaying for inhibitors of Aβproduction in a cell culture system consisting of a first transfectedcell type expressing β-APP but no PS mixed with a second cell typeexpressing PS but no β-APP. The inhibitory effect of an agent added tothis mixed cell culture would be measured from the activities of severallikely downstream effects of (a) the G_(oA) and G_(oB) interaction withPS-1 and PS-2; or (b) the Src family of tyrosine kinases; or (c) theinteraction of N-terminal domain of βAPP with the N-terminal domain ofPS-1 and/or PS-2.

In another aspect, the invention provides a method of improvingcognitive function and/or memory in a subject. The method includesadministering an agent that inhibits the interaction of PS-1 and/or PS-2with G-protein, G_(oA) and G_(oB). In one approach, the agent interactswith the C-terminal tail and/or other cytoplasmic domains of PS-1 and/or2 that interact with G_(oA) and/or G_(oB). The agent may also interferewith the downstream results of PS-1 and/or PS-2 binding to G_(o) such asG_(o) activation with phospholipase C. In another approach, the agentinhibits the activity of members of the Src family of tyrosine kinasesin cells expressing PS-1 and/or PS-2. In each case the agent would beadministered in an amount to improve cognitive function and/or memoryretention compared to a control subject.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a representative study to determine if PS-1 is a GPCR.Extracts of different cell cultures were analyzed in order to determinewhether G_(o) interacts with PS-1, including the necessary controls. Ineach lane, the particular cell extracts were first immunoprecipitatedwith a monoclonal Ab (MAb) directed to PS-1; the immunoprecipitate wasthen dissolved and subjected to SDS-PAGE electrophoresis, and theresulting gel was Western blotted with an antibody directed to G_(o)(this antibody recognizes both G_(oA) and G_(oB)) Lane 1 is a control ofan extract of untransfected ES (PS-1^(−/−)/PS-2^(−/−)) cells. Asexpected, this extract showed that no G_(oA) (or G_(oB)) wasimmunoprecipitated with Ab to PS-1. Lane 2 is an extract of ES cells,that had first been transfected with PS-1 only, but not with G_(oA). Noprotein band was observed for G_(oA); this was another controlexperiment. Lane 3 is an extract of the ES cells transfected with bothPS-1 and G_(oA). In this extract, G_(oA) is immunoprecipitated alongwith the PS-1, showing that PS-1 was bound to G_(oA), but not G_(oB). IfPS-1 without its C-terminal “tail” (lane 4), which protrudes from themembrane into the aqueous intracellular compartment), is transfectedinto ES double null cells along with G_(oA) (lane 6), little or noG_(oA) is immunoprecipitated along with the PS-1 tailess, showing thatthe C-terminal domain of PS-1 is the principal region of G_(oA) bindingto PS-1.

FIG. 2 shows a Western blot of a similar experiment to that of FIG. 1but with PS-2 instead of PS-1. Lanes 2 and 4 show that tail-less PS-2,unlike tail-less PS-1, still binds G_(oA) (and G_(oB)), and thereforethat the binding sites for G_(oA) and G_(oB) are not confined to theC-terminal domain of PS-2, as is the case for PS-1 (FIG. 1) Lane 1 isuntransfected ES (PS-1^(−/−)/PS-2^(−/−)). Lane 2 is PS-2+G_(oA). Lane 3is Tail-less PS-2+G_(oA). Lane 4 is PS-2+G_(oB). Lane 5 is Tail-lessPS-2+G_(oB).

FIG. 3 involves an independent way of demonstrating G_(oA) binding toPS-1. [³⁵S]-GTPγS, an analog of GTP, makes a covalent bond to the activesite of a G-protein, that is blocked by a prior reaction with Pertussistoxin (PTx). In lane 2, there is shown an 8-fold increase in³⁵S-incorporation into G_(oA) that is immunoprecipitated with antibodyto PS-1, but not into G_(oB) (lane 4). Therefore, PS-1 binds to G_(oA)(that has reacted with [³⁵S]-GTPγS to identify it as a G-protein (lane2), but also to a lesser extent to GOB than to G_(oA) (lane 4). The ³⁵Sbindings to G_(oA) and G_(oB) are blocked by prior treatment with PTx(lanes 3 and 5).

FIG. 4 is a graph depicting ³⁵SGTPγS incorporation in extracts of EScells transfected with cDNA for PS-2 and G-protein G_(oA).

The following two experiments are designed to determine if mouse PS is aGPCR in vivo in the normal mouse brain.

FIG. 5 shows the ³⁵S-GTPγS incorporation in extracts of mouse brain thatcould be immunoprecipitated with monoclonal antibodies to PS-1.

FIG. 6 shows the ³⁵S-GTPγS incorporation in extracts of mouse brain thatcould be immunoprecipitated with monoclonal antibodies to PS-2.Therefore, endogenous PS-1 and PS-2 in mouse brain are GPCRs.

FIGS. 5A-E shows immunofluorescence microscopic labeling of fixed cells,a) Double immunofluorescence microscopic labeling of untransfected,fixed but not permeabilized, DAMI cells with primary rat Mab #1563 tohuman PS-1 N-terminal domain (Panel 1) and FITC conjugated anti-rat IgGsecondary antibody shows cell-surface immunolabeling of endogenous PS-1amino terminal domain. Panel 2 shows the same cells do not expressappreciable amounts of cell-surface β-APP when labeled with Mab #348 tothe β-APP extracellular domain and TRITC-conjugated anti-mouse IgGsecondary antibody. Panel 3 shows the Nomarski images of cells in panels1 and 2. b) Double Immunofluorescence microscopic labeling ofβ-APP-transfected, fixed but not permeabilized, DAMI cells showscell-surface expressed β-APP when labeled with Mab #348 to the β-APPextracellular domain and TRITC-conjugated secondary antibody (Panel 2).Panels 1 and 3, the same cells treated as for FIG. 5 a. c)Immunofluorescence microscopic labeling of PS-1-transfected, fixed butnot permeabilized, DAMI cells shows high expression of cell-surface PS-1(Panel 1) but not β-APP (Panel 2) when labeled with the same primary andsecondary antibodies described in a. Panel 3 shows the Nomarski image ofcells in panels 1 and 2. These experiments show that transfection of theDAMI cells with PS-1 does not call forth cell surface expression ofβ-APP. d) Immunofluorescence microscopic labeling of β-APP-transfected,fixed but not permeabilized ES cells, double-null for PS-1 and PS-2.Cells show cell-surface expressed β-APP when labeled with Mab #348 tothe β-APP extracellular domain and TRITC-conjugated secondary antibody(Panel 2). Panel 1 shows the result of labeling with primary rat Mab#1563 to human PS-1 N-terminal domain and FITC conjugated appropriatesecondary antibody, indicating the expected absence of PS-1 on thesurfaces of ES double-null cells. Panel 3 shows Nomarski image of cellsin Panels 1 and 2. e) Immunofluorescence microscopic labeling ofuntransfected, fixed but not permeabilized ES cells, double-null forPS-1 and PS-2. Cells show cell-surface expressed endogenous mouse β-APPwhen labeled with Mab #348 to the β-APP extracellular domain andTRITC-conjugated secondary antibody (Panel 2). Panels 1 and 3 labeled asin d; no cell surface labeling for PS-1 (Panel 1) is observed in theseuntransfected ES cells. Bar, 20 μm.

FIG. 6 shows that within minutes after mixing β-APP-only expressingtransfected ES cells with PS-1 only expressing transfected DAMI cells, atransient protein tyrosine phosphorylation process arises in the mixedcell culture, as detected by ELISA analyses of the cell extracts. Thisactivity peaked at ˜8-10 mins after mixing (a). The same experimentcarried out in the presence of 25 μg purified soluble β-APP (b) or 25 μgpurified peptide of N-terminal domain of PS-1 fused to FLAG (c) showednone of the increases observed in (a). The addition of 25 μg of purifiedpeptide of the non-specific N-terminal domain of PS-2 fused to FLAG (d),however, resulted in very similar transient increases in proteintyrosine kinase activity to (a).

FIG. 7A-D show Experiments to determine the nature of the tyrosinephosphorylating enzyme activity in FIG. 6. Src family kinase assay withsynthetic peptides. a and b: β-APP:PS-1 interaction with separatelytransfected DAMI cells as a function of time after mixing. Src kinaseactivity was assayed using the Src family substrate peptide{lys19}cdc2(6-20)-NH2 (black bars) and control peptides {lys19Phe15}cdc2(6-20)NH2 (white bars) and {lys19ser14val12}cdc2(6-20)NH2 (graybars) for both the β-APP:PS-1 (a) and control pcDNA3:PS-1 (b)interactions. c and d: β-APP:PS-2 interaction with separatelytransfected DAMI cells as a function of time after mixing. Src kinaseactivity was assayed using the Src family substrate peptide{lys19}cdc2(6-20)-NH2 (black bars) and control peptides {lys19Phe15}cdc2(6-20)NH2 (white bars) and {lys19ser14val12}cdc2(6-20)NH2 (graybars) for both the β-APP:PS-2 (c) and control pcDNA3:PS-2 (d)interactions.

FIG. 8A-B shows Inhibition of tyrosine kinase activity. ELISAs todemonstrate tyrosine kinase activity of DAMI cells which had beenseparately transfected with β-APP and PS-1 and mixed in the presence andabsence of 10 μg/ml Herbimycin A (a) and 10 nM PP2 (b), as a function oftime after mixing.

FIG. 9A-B shows β-APP:PS-1 intercellular interaction: C-Src activity inextracts of mixed cells. a. Western Immunoblot. β-APP:PS-1 interactionswith mixtures of separately transfected DAMI cells. Western immunoblotwith primary anti-PTyr polyclonal antibodies (Panel 1) andanti-pp60c-src monoclonal antibodies (Panel 2) from the same experimentin which β-APP-transfected DAMI cells were mixed with PS-1-transfectedDAMI cells for 0-12 mins. Panel 3: Antibody labeling of controlpp60c-src protein with the pp60c-src antibodies. Panel 4: Westernimmunoblots with primary anti-PTyr antibodies, as in Panel 1, fromexperiments in which β-APP-transfected ES double-null cells wereinteracted with PS-1-transfected DAMI cells. b. Autoradiograph ofin-vitro phosphorylated proteins. Extracts of separately transfectedβ-APP and PS-1 DAMI cell mixtures at 0-12 mins after mixing were firstimmunoprecipitated with antibodies to c-Src and then phosphorylated invitro with γ32P-ATP. Autophosphorylation reactions were subjected toSDS-PAGE followed by autoradiography.

FIG. 10A-B shows β-APP:PS-2 intercellular interaction: C-Src activity inextracts of mixed cells. a. Western Immunoblot. β-APP:PS-2 interactionin extracts of separately transfected and mixed DAMI cells as a functionof time after mixing. Panels 1 and 2: Same as FIG. 9 a except thatPS-2-transfected DAMI cells replaced PS-1-transfected cells in theintercellular interaction with β-APP and cells were mixed from 1-20mins. b. Autoradiograph of in-vitro phosphorylated proteins. Sameextracts as in part a. Same as 5b except that PS-2-transfected DAMIcells replaced PS-1-transfected DAMI cells in the intercellularinteraction with β-APP.

FIG. 11A-D shows β-APP:PS-2 intercellular interaction: Activity of Lynand Fyn in extracts of mixed cells. a and b. Western Immunoblots:β-APP:PS-2 interaction. Western immunoblot with primary anti-Lynpolyclonal antibodies (a, Panel 1) and anti-Fyn polyclonal antibodies(b, Panel 1) from the same experiment in which β-APP-transfected DAMIcells were mixed with PS-2-transfected DAMI cells for 0-20 mins andextracts made. No change with time in concentration of either Lyn or Fynprotein was observed. Panel 2: Antibody labeling of control Lyn (a) andFyn (b) protein with their respective antibodies. c and d.Autoradiograph of in-vitro phosphorylated proteins: β-APP:PS-2interaction. Extracts of mixtures of β-APP and PS-2 mixed transfectedcells at 0-20 mins after mixing were first immunoprecipitated withantibodies to Lyn (c) or Fyn (d) and then phosphorylated in vitro withγ32P-ATP. Autophosphorylation reaction products were subjected toSDS-PAGE followed by autoradiography.

FIG. 12 illustrates intracellular domains of PS.

FIG. 13 shows the effect of intercellular β-APP:PS interactions on Aβproduction.

DETAILED DESCRIPTION

As used herein, the singular forms “a,” “and,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a protein” includes a plurality of such proteinsand reference to “the cell” includes reference to one or more cellsknown to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The invention is based, in part, upon the interaction of variousG-protein coupled receptors (GPCRs), as well as their downstreameffectors, protein kinase activity and calcium homeostasis. GPCRscomprise one of the largest gene families in the human genome, andmediate a huge variety of cellular functions regulated byneurotransmitters, hormones, chemokines, and many other molecules.Timely uncoupling of GPCR signaling is crucial for maintainingappropriateness and integrity of the GPCR-mediated physiologicalfunctions. This uncoupling is primarily mediated by a much smaller genefamily, currently numbering seven members of GPCR kinases (GRKs). Thespecificity for a few GRK members to regulate a huge numbers of GPCRs iscontrolled in an agonist-dependent manner. In another words, GRKspreferentially bind to and phosphorylate agonist-occupied GPCRs touncouple receptor from corresponding G-protein, a process known ashomologous desensitization. Based on structural similarities, sevenknown GRK members are classified into four subfamilies (GRK1, GRK2/3,GRK4/5/6 and GRK7), with GRK2/3 and GRK5/6 having ubiquitousdistributions including brain. Dysregulation of GRK2, probably GRK5 aswell, has been implicated in the pathogenesis of chronic heart failure,myocardial ischemia, and hypertension, and other cardiovasculardisorders, where the GRKs have been extensively studied. Failure todesensitize rhodopsin signaling by GRK1 can lead to photoreceptor celldeath, and is believed to contribute to retinitis pigmentosa. Inaddition, increased GRK2 levels have been associated with opiateaddiction. Aside from these, however, roles of GRKs in many otherpathological conditions potentially associated with GPCR deregulation,such as in AD, remain virtually unexplored.

Due to the membrane location of GPCRs, GRK's retention on the plasmamembrane or in the cytosol physically affects its access and binding toGPCRs. In resting cells, GRK4 subfamily members (including GRK4/5/6) aretightly associated with the plasma membrane (Reference 10), while GRK2subfamily members (GRK2/3) are primarily cytosolic and translocate tothe membrane when cells are stimulated by GPCR agonists. However, inactive cells, subcellular localization of GRKs appears to be determinedby the content and capacity of GRK-binding factors in membrane versuscytosol. Phospholipids, particularlyphosphatidylinositol-4,5-biphosphate, appear to play a role in GRKsadherence to the membrane and bind GPCRs, while phosphatidylserine (PS)may also enhance GRK2 binding to GPCRs on the membrane. On the otherhand, calcium/calmodulin and other calcium-binding proteins, as well asactin, actinin, and the like may contribute to sequester GRKs in thecytosol and inhibit binding of GRKs to GPCRs.

In AD brains, significant membrane alterations, aberrantphosphoinositide metabolism, disrupted calcium homeostasis anddisorganized cytoskeleton proteins could all influence the subcellulardistribution of GRKs. In addition, increased β-amyloid, a hydrophobicpeptide central to AD pathogenesis, has been shown to decrease membranephosphatidylinositol-4,5-biphosphate and increase [Ca²⁺]_(i).

Evidence of a 7-TM structure (like that of rhodopsin) for PS-1 and PS-2has led to the examination regarding whether PS-1 and PS-2 belong to theG-Protein coupled receptor superfamily of proteins, which all shareessentially a similar structure. Although PS does not exhibit anysubstantial amino acid homologies with any of the approximately 1,000GPCR's so far examined, the fact that all of these GPCR's are 7-TMintegral proteins, with many showing no sequence homologies with anyothers, allows for the possibility that PS molecules are also GPCR's.GPCR activity of PS was identified using a N141I-PS-2 mutation. Themutation, linked with FAD in Volga German families, caused PC-12 celldeath in a Pertussis toxin (PTx) sensitive manner. Other studiessuggested that within the 39 amino acid residue carboxyl-terminal domainof PS-1 (located in the cytoplasm in almost all topographic models ofPS-1 in the membrane) there exists a specific binding and regulatingdomain for the brain G_(o) protein. This domain of PS-1 that binds G_(o)in vitro also shows some local amino acid sequence homologies with theG-binding domains of two other GPCR proteins, the D2-dopaminergic, andthe 5HT-1B receptors, as well as the G-protein activating oligopeptide,mastoparan. The possibility that PS-1 may be a functional GPCR isfurther described herein.

The present disclosure demonstrates that G-protein G_(o) bindsfull-length PS-1, and is inhibited by Pertussis toxin. In addition, onlyG_(oA) binds PS-1, not G_(oB). Transfection of ES null cells with atail-less construct of PS-1, demonstrates that most of the bindingoccurs at the carboxyl terminal tail of PS-1. However, these resultsalso indicate that other cytoplasmic loop regions may be involved in thebinding, since very small amounts of binding occurred in the presence oftail-less PS-1. The disclosure also demonstrates that the G-proteinbinds not only to PS-1 but also PS-2 and that for PS-2, in addition tothe binding of G_(oA), G_(oB) also binds intact PS-2, not seen for PS-1.This binding is still present when tail-less PS-2 is used in place offull-length PS-2. These results suggest that G_(oB) binds PS-2 at acytoplasmic domain other than the C-tail. A greater than 700% increasein ³⁵S-GTPγS— labeled Gα_(oA) (but not Gα_(oB)) binding to PS-1. ForPS-2 there is similarly a greater than 700% increase over basal levelsof ³⁵S-GTPγS— labeled Gα_(oA) binding as well as ˜300% increase in³⁵S-GTPγS-labeled Gα_(oB). Treatment with PTx inhibits the incorporationof ³⁵S-GTPγS to both G_(oA) and G_(oB).

Thus, G_(oA) appears to bind both PS-1 and PS-2 at similar rates,whereas the binding of G_(oB) to PS-2 is less than half that observedfor G_(oA) under the same experimental conditions. The data confirm afunctional consequence of the G-protein coupling to PS-1 and PS-2 andfurther characterize the two presenilin proteins as G-protein coupledreceptors (GPCRs).

GPCRs have been classified into three main families according to theirsequence homology and structural features. Family 1 is the largest,constituting 90% of all GPCRs. Members of this family have a short aminoterminal extracellular domain and several conserved amino acid motifswithin the 7-TM domain. A “signature” of family 1 GPCRs is a conservedtripeptide DRY sequence at the interface of TM-III and 2nd intracellularloop that plays a critical role in G-protein coupling. PS-1 and PS-2have none of the conserved family 1 motifs, including the DRY sequence,and are unlikely to belong to this group. Members of family 2 share alonger extra-cellular domain and are activated by large peptide ligandssuch as glucagons and secretin. Members of family 3 include themetabotropic glutamate receptors (mGluRs), γ-aminobutyric acid B(GABA_(B)) receptors and the extra-cellular cation-sensing Ca²⁺ receptorand have large extra-cellular domains that function as ligand-bindingdomains. It is thought that this family utilizes distinct intracellulardomains and mechanisms for G-protein signaling. The conserved amino acidmotifs and DRY sequence present in family 1 GPCRs are not conserved infamily 3 and it is thought that the molecular events that lead to aconformational change in the proteins are therefore somewhat distinctbetween members of family 1 and family 3 GPCRs.

PS-1 and PS-2 appear to have more features in common with family 3 GPCRsthan with either of the other two families—both have largeextra-cellular domains (the N-terminal, and the hydrophilic loop betweenTM VI and VII), a feature of family 3 GPCRs. Ligand binding in family 3GPCRs appears to take place exclusively via the extra-cellular domains,generally the amino terminal domain. The N-terminal domain of PS-1 orPS-2 is sufficient for in vitro binding of PS-1 or PS-2 respectively, toβ-APP, a proposed ligand and possible agonist of PS GPCR activation.Some family 3 members form homodimers, usually by di-sulfide bonds viaextra-cellular Cys residues. It is well known that PS-1 and PS-2 existin the membrane as dimers. Further, they both have Cys residues in theirextra-cellular domains (7-TM structure), although it is not knownwhether these form di-sulfide bonds and participate in dimerization ofthe proteins. Family 3 GPCRs all have the 3rd intracellular loop as theshortest loop and this is conserved among each type. Likewise, the thirdintracellular loop in PS-1 and PS-2 is the shortest loop, consisting ofthe sequence KYLPEW (SEQ ID NO:1), which is completely conserved. Somemembers of family 3 GPCRs interact directly via their carboxyl terminalPDZ binding domains with intracellular PDZ-domain proteins such asHomer. There is a PDZ binding domain in the carboxyl terminal tail ofPS-1 which has been shown to bind to several PDZ proteins.

Studies on the mechanisms of Aβ production have involved cell-cellinteraction of β-APP on the surface of one cell with PS-1 or PS-2 on thesurface of another cell. The invention suggests that the 8-amyloidprecursor protein β-APP and PS-1 or 2 may normally be components of anintercellular signaling system. One or more forms of β-APP canspecifically bind either to PS-1, or PS-2, via their extracellulardomains that protrude from their respective cell membranes. This bindingin vivo induces an intercellular signaling event of significance tonormal neural physiology or development. A by-product of thistranscellular molecular binding, processes of vesicle formation,cellular internalization, and proteolytic degradation are set in motionthat result in the formation and cellular release of Aβ and its slowaccumulation in regions of the brain.

PS are expressible at the cell-surface and have 7-TM structures and PS-1and PS-2 participate in a specific cell-cell interaction with β-APP;this β-APP:PS mediated intercellular interaction results in transientincrease in tyrosine kinase activity and protein tyrosinephosphorylation. Furthermore, a β-APP:PS mediated cell-cell interactionis required for at least the major part of the production of Aβ. Theintercellular interaction between β-APP and PS may also activateG-protein binding to PS. (There is now substantial evidence that thereis cross-talk between protein tyrosine kinases and the G-proteinsignaling pathways).

If G_(o) activation by PS ultimately affects Aβ production, then apossible outcome of these last studies might be a drug therapy for ADusing appropriately designed inhibitors of PS-G_(o) specific binding.

Thus, it appears that PS-1, PS-2 and APP play a role in intracellularsignaling. The primary focus of investigations of these three proteinshas been on their respective roles in the proteolytic fragmentation ofβ-APP to Aβ that involves the PS proteins either directly or indirectly.In addition, one or more forms of β-APP on one cell surface and PS-1 (orPS-2) on another may be specific ligand and receptor components of anintercellular signaling system with a role in normal physiology. Thedisclosure provides evidence that intercellular surface binding of β-APPto the PS proteins functions in normal physiology to induce a signalingprocess within one, or possibly both, of the adherent cells, leadingultimately to a developmental outcome significant for the organism.

This proposal was based on an analogous intercellular signaling betweenpre-R7 and R8 cells in the development of the Drosophila eye. InDrosophila, the Type I single-TM spanning protein Sevenless (SEV)(similar to β-APP) on the pre-R7 cell surface binds specifically to the7-TM Bride of Sevenless (BOSS) protein (similar to PS-1 or PS-2). Asdescribed elsewhere herein, the PS proteins are of 7-TM topography, not8, as widely accepted. In this case, signaling requires that thetyrosine kinase activity of the cytoplasmic domain of the SEV protein beactivated. Neither B-APP nor the PS proteins are protein tyrosinekinases and if protein tyrosine phosphorylation were involved, anotherindirect activity of the cytoplasmic domain(s) would, in this case, haveto provide the downstream signal. Experiments were initially undertakento detect such possible intercellular protein tyrosine phosphorylationsignaling events. It was shown that when cultured DAMI (humanmegakaryoblast) cells that were transiently transfected with β-APP weremixed with DAMI cells transfected with PS-1, or PS-2, within severalminutes after mixing, the cell extracts showed significant transientincreases in protein tyrosine kinase activity and in phosphotyrosine(PTyr) modification of protein substrates, that did not appear incontrols, or in cell mixtures containing inhibitors of the specificβ-APP:PS binding. The downstream consequences of this signaling weredifferent depending on whether PS-1 or PS-2 was engaged in theintercellular binding to β-APP, because the spectrum of proteins thatshowed enhanced tyrosine phosphorylation was altogether different in thetwo cases, suggesting a distinction between, rather than a redundancyof, the biochemical functions of the two closely homologous PS proteins.

The disclosure demonstrates the biological pathways by using embryonicstem (ES) cells derived from PS-1^(−/−), PS-2^(−/−) double null miceherein referred to as ES double-null cells, either untransfected incontrol experiments, or transfected with β-APP. In the latter case, theβ-APP-transfected ES cells are mixed with either PS-1- orPS-2-transfected DAMI cells; the DAMI cells do not express significantamounts of endogenous β-APP on their surfaces. In this mixedcell-culture system, therefore, the β-APP-transfected ES double-nullcells serve as the only source of cell-surface expressed β-APP, whilethe PS-transfected DAMI cells are the only source of cell-surfaceexpressed PS. If a β-APP:PS specific signaling event occurs in thissystem, it can be the result of a juxtacrine interaction between the twocell types. In the present study, such an interaction has been found.

The increase in PTyr protein modification that is the consequence ofβ-APP:PS intercellular binding involves a protein tyrosine kinase(s) tobe determined. Evidence is provided that signaling is accompanied bytransient elevations in Src family tyrosine kinase activity, and hasidentified the individual Src family member mediating the intercellularsignaling between β-APP and PS-1, but not PS-2, to be pp60c-src. Incontrast, the β-APP:PS-2 signaling involves the Src family member Lyn.These signaling events affect normal physiology. For example, they mayplay a role in the physiological defects encountered in the developmentof β-APP null mice.

The Src family of kinases are implicated in cancer, immune systemdysfunction and bone remodeling diseases. For general reviews, seeThomas and Brugge, Annu. Rev. Cell Dev. Biol. 1997, 13, 513; Lawrenceand Niu, Pharmacol. Ther. 1998, 77, 81; Tatosyan and Mizenina,Biochemistry (Moscow) 2000, 65, 49-58; Boschelli et al., Drugs of theFuture 2000, 25(7), 717.

Members of the Src family include the following eight kinases inmammals: Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and Blk. These arenonreceptor protein kinases that range in molecular mass from 52 to 62kD. All are characterized by a common structural organization that iscomprised of six distinct functional domains: Src homology domain 4(SH4), a unique domain, SH3 domain, SH2 domain, a catalytic domain(SH1), and a C-terminal regulatory region. Tatosyan et al. Biochemistry(Moscow) 2000, 65, 49-58.

Based on published studies, Src kinases are considered as potentialtherapeutic targets for various human diseases. Mice that are deficientin Src develop osteopetrosis, or bone build-up, because of depressedbone resorption by osteoclasts. This shows that osteoporosis resultingfrom abnormally high bone resorption is treated by inhibiting Src.Soriano et al., Cell 1992, 69, 551 and Soriano et al., Cell 1991, 64,693.

Suppression of arthritic bone destruction has been achieved by theoverexpression of CSK in rheumatoid synoviocytes and osteoclasts.Takayanagi et al., J. Clin. Invest. 1999, 104, 137. CSK, or C-terminalSrc kinase, phosphorylates and thereby inhibits Src catalytic activity.This implies that Src inhibition may prevent joint destruction that ischaracteristic in patients suffering from rheumatoid arthritis.Boschelli et al., Drugs of the Future 2000, 25(7), 717.

Src also plays a role in the replication of hepatitis B virus. Thevirally encoded transcription factor HBx activates Src in a steprequired for propagation of the virus. Klein et al., EMBO J. 1999, 18,5019, and Klein et al., Mol. Cell. Biol. 1997, 17, 6427.

A number of studies have linked Src expression to cancers such as colon,breast, hepatic and pancreatic cancer, certain B-cell leukemias andlymphomas. Talamonti et al., J. Clin. Invest. 1993, 91, 53; Lutz et al.,Biochem. Biophys. Res. 1998 243, 503; Rosen et al., J. Biol. Chem. 1986,261, 13754; Bolen et al., Proc. Natl. Acad. Sci. USA 1987, 84, 2251;Masaki et al., Hepatology 1998, 27, 1257; Biscardi et al., Adv. CancerRes. 1999, 76, 61; Lynch et al., Leukemia 1993, 7, 1416. Furthermore,antisense Src expressed in ovarian and colon tumor cells has been shownto inhibit tumor growth. Wiener et al., Clin. Cancer Res., 1999, 5,2164; Staley et al., Cell Growth Diff. 1997, 8, 269.

Other Src family kinases are also potential therapeutic targets. Lckplays a role in T-cell signaling. Mice that lack the Lck gene have apoor ability to develop thymocytes. The function of Lck as a positiveactivator of T-cell signaling suggests that Lck inhibitors may be usefulfor treating autoimmune disease such as rheumatoid arthritis. Molina etal., Nature, 1992, 357, 161. Hck, Fgr and Lyn have been identified asimportant mediators of integrin signaling in myeloid leukocytes. Lowellet al., J. Leukoc. Biol., 1999, 65, 313. Inhibition of these kinasemediators may therefore be useful for treating inflammation. Boschelliet al., Drugs of the Future 2000, 25(7), 717.

GSK-3 activity is also associated with Alzheimer's disease. This diseaseis characterized by the presence of the well-known β-amyloid peptide andthe formation of intracellular neurofibrillary tangles. Theneurofibrillary tangles contain hyperphosphorylated Tau protein, inwhich Tau is phosphorylated on abnormal sites. GSK-3 has been shown tophosphorylate these abnormal sites in cell and animal models.Furthermore, inhibition of GSK-3 has been shown to preventhyperphosphorylation of Tau in cells [Lovestone et al., Curr. Biol., 4,1077-86 (1994); and Brownlees et al., Neuroreport 8, 3251-55 (1997);Kaytor and Orr, Curr. Opin. Neurobiol., 12, 275-8 (2000)]. In transgenicmice overexpressing GSK3, significant increased Tau hyperphosphorylationand abnormal morphology of neurons were observed (Lucas et al., EMBO J,20:27-39 (2001)). Active GSK3 accumulates in cytoplasm of pretangledneurons, which can lead to neurofibrillary tangles in brains of patientswith AD (Pei et al., J Neuropathol Exp Neurol, 58, 1010-19 (1999)).Therefore, inhibition of GSK-3 slows or halts the generation ofneurofibrillary tangles and thus treats or reduces the severity ofAlzheimer's disease.

Evidence for the role GSK-3 plays in Alzheimer's disease has been shownin vitro. See Aplin et al. (1996), J Neurochem 67:699; Sun et al.(2002), Neurosci Lett 321:61 (GSK3b phosphorylates cytoplasmic domain ofAmyloid Precursor Protein (APP) and GSK3b inhibition reduces Ab40 andAb42 secretion in APP-transfected cells); Takashima et al. (1998), PNAS95:9637; Kirschenbaum et al. (2001), J Biol Chem 276:7366 (GSK3bcomplexes with and phosphorylates presenilin-1, which is associated withgamma-secretase activity in the synthesis of Ab from APP); Takashima etal. (1998), Neurosci Res 31:317 (Activation of GSK3b by Ab(25-35)enhances phosphorylation of tau in hippocampal neurons. This observationprovides a link between Ab and neurofibrillary tangles composed ofhyperphosphorylated tau, another pathological hallmark of AD); Takashimaet al. (1993), PNAS 90:7789 (Blockade of GSK3b expression or activityprevents Ab-induced neuro-degeneration of cortical and hippocampalprimary cultures); Suhara et al. (2003), Neurobiol Aging. 24:437(Intracellular Ab42 is toxic to endothelial cells by interfering withactivation of Akt/GSK-3b signaling-dependent mechanism); De Ferrari etal. (2003) Mol Psychiatry 8:195 (Lithium protects N2A cells and primaryhippocampal neurons from Ab fibrils-induced cytotoxicity, & reducednuclear translocation/destabilization of β-catenin); and Pigino et al.,J Neurosci, 23:4499, 2003 (The mutations in Alzheimer's presenilin 1 mayderegulate and increase GSK-3 activity, which in turn, impairs axonaltransport in neurons. The consequent reductions in axonal transport inaffected neurons can ultimately lead to neurodegeneration).

Evidence for the role GSK-3 plays in Alzheimer's disease has been shownin vivo. See Yamaguchi et al. (1996), Acta Neuropathol 92:232; Pei etal. (1999), J Neuropath Exp Neurol 58:1010 (GSK3b immunoreactivity iselevated in susceptible regions of AD brains); Hernandez et al. (2002),J Neurochem 83:1529 (Transgenic mice with conditional GSK3boverexpression exhibit cognitive deficits similar to those in transgenicAPP mouse models of AD); De Ferrari et al. (2003) Mol Psychiatry 8:195(Chronic lithium treatment rescued neurodegeneration and behavioralimpairments (Morris water maze) caused by intrahippocampal injection ofAb fibrils.); McLaurin et al., Nature Med, 8:1263, 2002 (Immunizationwith Ab in a transgenic model of AD reduces both AD-like neuropathologyand the spatial memory impairments); and Phiel et al. (2003) Nature423:435 (GSK3 regulates amyloid-beta peptide production via directinhibition of gamma secretase in AD tg mice).

Presenilin-1 and kinesin-1 are also substrates for GSK-3 and relate toanother mechanism for the role GSK-3 plays in Alzheimer's disease, aswas recently described by Pigino, G., et al., Journal of Neuroscience(23:4499, 2003). It was found that GSK3beta phosphorylates kinsesin-1light chain, which results in a release of kinesin-1 from membrane-boundorganelles, leading to a reduction in fast anterograde axonal transport.A mutations in PS-1 may deregulate and increase GSK-3 activity, which inturn, impairs axonal transport in neurons. The consequent reductions inaxonal transport in affected neurons ultimately lead toneurodegeneration.

The invention supports that specific adhesion between β-APP-presentingand PS-presenting cells might have different physiological consequences,one a transcellular (juxtacrine) signaling process associated with thenormal function of these proteins, and the other resulting eventually inthe proteolysis of β-APP to form Aβ, leading to the pathology ofAlzheimer's disease. Evidence for a juxtacrine interaction in thissystem was obtained with cultured DAMI cells appropriately transfectedwith either β-APP, or with PS-1 or PS-2; a specific β-APP:PS mediatedcell-cell interaction led to rapid and transient increases in proteintyrosine kinase activity and protein tyrosine phosphorylation withinmost likely one, or possibly both, of the adhering cells. DAMI cellswere employed because these cells do not normally express significantamounts of endogenous β-APP at the cell surface, and because they areeasy to detach mechanically from the cell substratum. Thus, bytransfecting ES double-null cells with β-APP, cells expressing onlysurface β-APP but not PS were made available, and by transfecting DAMIcells with either PS-1 or PS-2, additional cells were produced thatexpressed a PS protein at the surface, and no significant β-APP.

Mixing experiments between these transfected cells, as the results show,clearly reveal signaling specifically between β-APP and PS (FIG. 5),which result from a juxtacrine interaction; i.e., a reaction involvingmembrane-bound PS on one cell surface with β-APP on another. Thisinteraction is specifically inhibited both by soluble β-APP (theexoplasmic domain of β-APP), and by the N-terminal domain of PS-1 fusedto FLAG, demonstrating the dual specificity of the interaction of β-APPwith PS.

The downstream consequences of this signaling are different depending onwhether PS-1 or PS-2 is engaged in the intercellular binding to β-APP.The spectrum of proteins modified by tyrosine phosphorylation differeddepending on whether PS-1 or PS-2 was involved in the specificintercellular binding to β-APP. Here the invention identifies c-Src as aprotein that undergoes the major transient increases in phosphorylationwhen β-APP and PS-1 interact intercellularly, yet it appears not to beinvolved in the increase in Src family kinase activity observed whenβ-APP undergoes cell-cell interaction with PS-2. For the latter, the Srckinase family member Lyn appears to be the predominant (or at least amajor) Src kinase involved. Together these results suggest distinctsignaling mechanisms that might result in different rather thanredundant physiological functions for the two closely homologouspresenilin proteins.

The invention demonstrates that juxtacrine signaling between β-APP andeither PS-1 or PS-2 results in rapid transient tyrosine kinaseactivation that are differentiable between the two PS proteins. However,none of these proteins are themselves tyrosine kinases, and some typesof indirect activation of Src family kinase activities seem to beinvolved. In general, members of the Src family of tyrosine kinases canbe activated indirectly by binding to receptors or other proteins usinga variety of different molecular mechanisms. Src tyrosine kinases can beregulated by binding to specific receptors and they in turn can regulatethe functional activity of the receptors. C-Src or Lyn may be recruitedupon the binding of β-APP with PS-1 or PS-2, respectively. Recruitmentwould suggest that a signaling complex is formed transiently in vivo atsites of cell-cell contact, setting in motion a cascade ofphosphorylation events that could result in developmental consequences.Identifying the region(s) of Src necessary for association with theβ-APP:PS-1 complex should yield valuable information regarding theassembly and activation of a β-APP:PS-1 signaling complex and shouldindicate whether or not the interaction between the β-APP:PS complex andthe kinases is direct or indirect. β-APP is not known to bephosphorylated on cytoplasmic tyrosine residues, and neither is PS-1, sodirect binding through the SH2 domain of c-Src is unlikely since thisdomain binds only at phosphorylated tyrosine residues.

Direct binding may however alternatively occur via the SH3 domain ofSrc. 5H3 domains recognize proline-rich sequences containing the coreP-X-X-P (SEQ ID NO:2), where X denotes any amino acid. Ligands recognizethe SH3 binding surface in one of two opposite orientations. Peptidesthat bind in a type 1 orientation conform to the consensus sequenceR-X-L-P-X-Z-P (SEQ ID NO:3) where Z is normally a hydrophobic or Argresidue (Kay et al., 2000). Interestingly, both PS-1 and PS-2 have aconserved type 1SH3 binding site (LPALP) in the cytoplasmic carboxylterminal region (residues 432-436 of PS-1 and residues 412-416 of PS-2),but the identity of these SH3 binding sites does not account for thedifferentiable activation of PS-1 and PS-2 by β-APP.

Tyrosine phosphorylation has been implicated in the regulation of avariety of biological responses and the tyrosine kinases involved inmediating these responses are made up of a diverse spectrum of proteins.Src kinases have been implicated in adhesion events regulated by thereceptors they bind and are activated following engagement of multiplereceptor pathways that regulate cell-cell and cell-matrix interactions.It seems likely that β-APP:PS signaling might play a significant role innormal developmental physiology. Whether such signaling is in addition arequired early step in the pathway of Aβ production, might beexperimentally investigated by determining the effect of inhibiting thejuxtacrine tyrosine phosphorylation on Aβ production.

A number of agents that inhibit GPCR interactions are known in the art.In addition, a number of kinase (e.g., c-src, fln and the like)inhibitors are known in the art and can be used in the methods of theinvention. Compositions comprising such agents in pharmaceuticallyacceptable carriers for treating AD are contemplated by the invention.

GPCRs share a common structural motif. Generally, these receptors haveseven sequences of between 22 to 24 hydrophobic amino acids that formseven alpha helices, each of which spans the membrane (each span isidentified by number, i.e., transmembrane-1 (TM-1), transmembrane-2(TM-2), etc.). The transmembrane helices are joined by strands of aminoacids between transmembrane-2 and transmembrane-3, transmembrane-4 andtransmembrane-5, transmembrane-6 and transmembrane-7 on the exterior, or“extracellular” side, of the cell membrane (these are referred to as“extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).The transmembrane helices are also joined by strands of amino acidsbetween transmembrane-1 and transmembrane-2, transmembrane-3 andtransmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when a ligand binds with the receptor (often referred to as“activation” of the receptor), there is a change in the conformation ofthe intracellular region that allows for coupling between theintracellular region and an intracellular “G-protein.” It has beenreported that GPCRs are “promiscuous” with respect to G proteins, i.e.,that a GPCR can interact with more than one G protein. See, Kenakin, T.,43 Life Sciences 1095 (1988). Although other G proteins exist,currently, Gq, Gs, Gi, Gz and Go are G proteins that have beenidentified. Endogenous ligand-activated GPCR coupling with the G-proteinbegins a signaling cascade process (referred to as “signaltransduction”). Under normal conditions, signal transduction ultimatelyresults in cellular activation or cellular inhibition. It is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Receptor activated G proteins are bound to the inside surface of thecell membrane. They consist of the Gα and the tightly associated Gβγsubunits. When a ligand activates the G protein-coupled receptor, itinduces a conformation change in the receptor (a change in shape) thatallows the G protein to now bind to the receptor. The G protein thenreleases its bound GDP from the Gα subunit, and binds a new molecule ofGTP. This exchange triggers the dissociation of the Gα subunit, the Gβγdimer, and the receptor. Both, Gα-GTP and Gβγ, can then activatedifferent signalling cascades (or second messenger pathways) andeffector proteins, while the receptor is able to activate the next Gprotein. The Gα subunit will eventually hydrolyze the attached GTP toGDP by its inherent enzymatic activity, allowing it to reassociate withGβγ and starting a new cycle.

The alpha subunit of the guanine nucleotide-binding protein G_(o) (“o”for other) mediates signal transduction between a variety of receptorsand effectors. Two forms of Go alpha subunit have been isolated frombrain tissue libraries. These two forms, G_(oA) alpha and G_(oB) alpha(also referred to as G_(oA) and G_(oB)), are the products of alternativesplicing. G_(oA) alpha transcripts are present in a variety of tissuesbut are most abundant in brain. The G_(oB) alpha transcript is expressedat highest levels in brain and testis.

Specific GPCR screening assay techniques are known to the skilledartisan. For example, once candidate compounds are identified using the“generic” G protein-coupled receptor assay (i.e., an assay to selectcompounds that are agonists, partial agonists, or inverse agonists),further screening to confirm that the compounds have interacted at thereceptor site is preferred For example, a compound identified by the“generic” assay may not bind to the receptor, but may instead merely“uncouple” the G protein from the intracellular domain.

G_(s) stimulates the enzyme adenylyl cyclase. G_(i) (and G_(z) andG_(o)), on the other hand, inhibit this enzyme. Adenylyl cyclasecatalyzes the conversion of ATP to cAMP; thus, constitutively activatedGPCRs that couple the G_(s) protein are associated with increasedcellular levels of cAMP. On the other hand, constitutively activatedGPCRs that couple G_(i) (or G_(z), G_(o)) protein are associated withdecreased cellular levels of cAMP. See, generally, “Indirect Mechanismsof Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3rd Ed.)Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus, assaysthat detect cAMP can be utilized to determine if a candidate compoundis, e.g., an inverse agonist to the receptor (i.e., such a compoundwould decrease the levels of cAMP). A variety of approaches known in theart for measuring cAMP can be utilized; a most preferred approach reliesupon the use of anti-cAMP antibodies in an ELISA-based format Anothertype of assay that can be utilized is a whole cell second messengerreporter system assay. Promoters on genes drive the expression of theproteins that a particular gene encodes. Cyclic AMP drives geneexpression by promoting the binding of a cAMP-responsive DNA bindingprotein or transcription factor (CREB) that then binds to the promoterat specific sites called cAMP response elements and drives theexpression of the gene. Reporter systems can be constructed which have apromoter containing multiple cAMP response elements before the reportergene, e.g., β-galactosidase or luciferase. Thus, a constitutivelyactivated Gs-linked receptor causes the accumulation of cAMP that thenactivates the gene and expression of the reporter protein The reporterprotein such as galactosidase or luciferase can then be detected usingstandard biochemical assays.

G_(q) and G_(o) are associated with activation of the enzymephospholipase C, which in turn hydrolyzes the phospholipid PIP₂,releasing two intracellular messengers: diacycloglycerol (DAG) andinistol 1,4,5-triphoisphate (IP₃). Increased accumulation of IP₃ isassociated with activation of G_(q)- and G_(o)-associated receptors.See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8,From Neuron To Brain (3^(rd) Ed.) Nichols, J. G. et al eds. SinauerAssociates, Inc. (1992). Assays that detect IP₃ accumulation can beutilized to determine if a candidate compound is, e.g., an inverseagonist to a G_(q)- or G_(o)-associated receptor (i.e., such a compoundwould decrease the levels of IP₃). G_(q)-associated receptors can alsobeen examined using an AP1 reporter assay in that G_(q)-dependentphospholipase C causes activation of genes containing AP1 elements;thus, activated Gq-associated receptors will evidence an increase in theexpression of such genes, whereby inverse agonists thereto will evidencea decrease in such expression, and agonists will evidence an increase insuch expression. Commercially available assays for such detection areavailable.

The term “agent” or “test compound” or “drug candidate” or “modulator”or grammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid, fattyacid, polynucleotide, RNAi or siRNA, asRNA, oligonucleotide, etc. Anagent is any molecule that can be tested in an assay to identify theabukity of the agent to modulate the activity of presenilin. The agentcan be in the form of a library of test compounds, such as acombinatorial or randomized library that provides a sufficient range ofdiversity. Agent are optionally linked to a fusion partner, e.g.,targeting compounds, rescue compounds, dimerization compounds,stabilizing compounds, addressable compounds, and other functionalmoieties. Conventionally, new chemical entities with useful propertiesare generated by identifying a test compound (called a “lead compound”)with some desirable property or activity, e.g., inhibiting activity,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. Often, high throughput screening(HTS) methods are employed for such an analysis.

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity, e.g., ligands, agonists, antagonists, andtheir homologs and mimetics. The term “modulator” includes inhibitorsand activators. Inhibitors are agents that bind to, partially or totallyblock stimulation or enzymatic activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity ofpresenilin, e.g., antagonists. Activators are agents that bind to,stimulate, increase, open, activate, facilitate, enhance activation orenzymatic activity, sensitize or up regulate the activity of presenilin,e.g., agonists. Modulators include naturally occurring and syntheticligands, antagonists, agonists, small chemical molecules and the like.Assays to identify inhibitors and activators include, e.g., applyingputative modulator compounds to cells, in the presence or absence ofpresenilin and then determining the functional effects on presenilinactivity. Samples or assays comprising presenilin that are treated witha potential activator, inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of effect. Control samples (untreated with modulators) areassigned a relative activity value of 100%. Inhibition is achieved whenthe activity value of presenilin relative to the control is about 80%,optionally 50% or 25-1%. Activation is achieved when the activity valueof presenilin relative to the control is 110%, optionally 150%,optionally 200-500%, or 1000-3000% higher.

An “agonist” refers to an agent that binds to a polypeptide orpolynucleotide of the invention, stimulates, increases, activates,facilitates, enhances activation, sensitizes or up regulates theactivity or expression of a polypeptide or polynucleotide of theinvention.

An “antagonist” refers to an agent that inhibits expression of apolypeptide or polynucleotide of the invention or binds to, partially ortotally blocks stimulation, decreases, prevents, delays activation,inactivates, desensitizes, or down regulates the activity of apolypeptide or polynucleotide of the invention.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 Daltons and less than about 2500 Daltons, preferably lessthan about 2000 Daltons, preferably between about 100 to about 1000Daltons, more preferably between about 200 to about 500 Daltons.

“Determining the functional effect” refers to assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of presenilin, e.g., measuring physical and chemicalor phenotypic effects of e.g., presenilin interactions with a G-proteinor β-APP. Such functional effects can be measured by any means known tothose skilled in the art, e.g., changes in spectroscopic (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the protein; measuringinducible markers or transcriptional activation of the protein;measuring binding activity or binding assays, e.g. binding toantibodies; measuring changes in ligand binding affinity; measurement ofcalcium influx; measurement of the accumulation of an enzymatic productof a polypeptide of the invention or depletion of an substrate; changesin enzymatic activity, measurement of changes in protein levels of apolypeptide of the invention; measurement of RNA stability; G-proteinbinding; GPCR phosphorylation or dephosphorylation; tau phosphorylationor dephosphorylation, signal transduction, e.g., receptor-ligandinteractions, second messenger concentrations (e.g., cAMP, IP₃, orintracellular Ca²⁺); identification of downstream or reporter geneexpression (CAT, luciferase, beta-gal, GFP and the like), e.g., viachemiluminescence, fluorescence, calorimetric reactions, antibodybinding, inducible markers, and ligand binding assays. In addition,λ-APP binding to presenilin and Aβ production can also be used asdeterminants of a functional effect on presenilin activity. The term“amyloid beta peptide” means amyloid beta peptides processed from theamyloid beta precursor protein (APP). The most common peptides includeamyloid beta peptides 1-40, 1-42, 11-40 and 11-42. Other less prevalentamyloid beta peptide species are described as x-42, whereby x rangesfrom 2-10 and 12-17, and 1-y whereby y ranges from 24-39 and 41. Fordescriptive and technical purposes, “x” has a value of 2-17, and “y” hasa value of 24 to 41.

Agents identified by methods provided herein may be administeredtherapeutically or prophylactically to treat diseases associated withamyloid fibril formation, aggregation or deposition, regardless of theclinical setting. The compounds of the invention may act to modulate thecourse of an amyloid related disease using any of the followingmechanisms, such as, for example but not limited to: slowing the rate ofamyloid fibril formation or deposition; lessening the degree of amyloiddeposition; inhibiting, reducing, or preventing amyloid fibrilformation; inhibiting amyloid induced inflammation; enhancing theclearance of amyloid from, for example, the brain; or protecting cellsfrom amyloid induced (oligomers or fibrillar) toxicity.

“Modulation” of amyloid deposition includes both inhibition, as definedabove, and enhancement of amyloid deposition or fibril formation. Theterm “modulating” is intended, therefore, to encompass prevention orstopping of amyloid formation or accumulation, inhibition or slowingdown of further amyloid aggregation in a subject with ongoingamyloidosis, e.g., already having amyloid aggregates, and reducing orreversing of amyloid aggregates in a subject with ongoing amyloidosis;and enhancing amyloid deposition, e.g., increasing the rate or amount ofamyloid deposition in vivo or in vitro. Amyloid-enhancing compounds maybe useful in animal models of amyloidosis, for example, to make possiblethe development of amyloid deposits in animals in a shorter period oftime or to increase amyloid deposits over a selected period of time.Amyloid-enhancing compounds may be useful in screening assays forcompounds which inhibit amyloidosis in vivo, for example, in animalmodels, cellular assays and in vitro assays for amyloidosis. Suchcompounds may be used, for example, to provide faster or more sensitiveassays for compounds. In some cases, amyloid enhancing compounds mayalso be administered for therapeutic purposes, e.g., to enhance thedeposition of amyloid in the lumen rather than the wall of cerebralblood vessels to prevent CAA. Modulation of amyloid aggregation isdetermined relative to an untreated subject or relative to the treatedsubject prior to treatment.

“Inhibition” of amyloid deposition includes preventing or stopping ofamyloid formation, e.g., fibrillogenesis, clearance of soluble Aβ frombrain, inhibiting or slowing down of further amyloid deposition in asubject with amyloidosis, e.g., already having amyloid deposits, andreducing or reversing amyloid fibrillogenesis or deposits in a subjectwith ongoing amyloidosis. Inhibition of amyloid deposition is determinedrelative to an untreated subject, or relative to the treated subjectprior to treatment, or, e.g., determined by clinically measurableimprovement, e.g., or in the case of a subject with brain amyloidosis,e.g., an Alzheimer's or cerebral amyloid angiopathy subject,stabilization of cognitive function or prevention of a further decreasein cognitive function (i.e., preventing, slowing, or stopping diseaseprogression), or improvement of parameters such as the concentration ofAβ or tau in the CSF.

As used herein, “treatment” of a subject includes the application oradministration of a composition comprising an agent identified by amethod of the invention to a subject, or application or administrationof a composition of the invention to a cell or tissue from a subject,who has a amyloid-β related disease or condition, has a symptom of sucha disease or condition, or is at risk of (or susceptible to) such adisease or condition, with the purpose of curing, healing, alleviating,relieving, altering, remedying, ameliorating, improving, or affectingthe disease or condition, the symptom of the disease or condition, orthe risk of (or susceptibility to) the disease or condition. The term“treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the subject; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating;improving a subject's physical or mental well-being; or, in somesituations, preventing the onset of dementia. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination or apsychiatric evaluation. For example, the methods of the inventionsuccessfully treat a subject's dementia by slowing the rate of or extentof cognitive decline.

While Alzheimer's disease of the familial or the sporadic type is themajor dementia found in the aging population, other types of dementiaare also found. These include but are not limited to: thefronto-temporal degeneration associated with Pick's disease, vasculardementia, senile dementia of Lewy body type, dementia of Parkinsonismwith frontal atrophy, progressive supranuclear palsy and corticobasaldegeneration and Downs syndrome associated Alzheimers'. Plaque formationis also seen in the spongiform encephalopathies such as CJD, scrapie andBSE. The present invention is directed to treatment of suchneurodegenerative diseases, particularly those involving neurotoxicprotein plaques, eg. amyloid plaques.

Downs syndrome is a serious human disorder that occurs with an incidenceof 1 in 800 live births. It is associated with the presence in affectedindividuals of an extra copy of chromosome 21 (trisomy 21). Theβ-amyloid precursor protein (β-APP) gene is encoded on chromosome 21,very close to the Down syndrome locus. All patients with Downs syndrome,if they survive beyond 40 years, develop Alzheimer's-like dementia andthe deposition of Aβ in their brains. There is good reason, therefore,to propose that the over-production of Aβ is connected directly with theoccurrence of the dementia in both AD and Downs syndrome. Therefore, thenature of the identification of therapeutic agents for the ameliorationof the symptoms of AD will also be useful for the amelioration of thesymptoms of Downs syndrome.

“Dementia” refers to a general mental deterioration due to organic orpsychological factors; characterized by disorientation, impaired memory,judgment, and intellect, and a shallow labile affect. Dementia hereinincludes vascular dementia, ischemic vascular dementia (IVD),frontotemporal dementia (FTD), Lewy body dementia, Alzheimer's dementia,etc. The most common form of dementia among older people is Alzheimer'sdisease (AD).

The expressions “mild-moderate” or “early stage” AD are used as synonymsherein to refer to AD which is not advanced and wherein the signs orsymptoms of disease are not severe. Subjects with mild-moderate or earlystage AD can be identified by a skilled neurologist or clinician. In oneembodiment, the subject with mild-moderate AD is identified using theMini-Mental State Examination (MMSE). Herein, “moderate-severe” or “latestage” AD refer to AD which is advanced and the signs or symptoms ofdisease are pronounced. Such subjects can be identified by a skilledneurologist or clinician. Subjects with this form of AD may no longerrespond to therapy with cholinesterase inhibitors, and my have amarkedly reduced acetylcholine level. In one embodiment, the subjectwith moderate-severe AD is identified using the Mini-Mental StateExamination (MMSE). “Familial AD” is an inherited form of AD caused by agenetic defect. A “symptom” of AD or dementia is any morbid phenomenonor departure from the normal in structure, function, or sensation,experienced by the subject and indicative of AD or dementia.

An agent may be administered therapeutically or prophylactically totreat diseases associated with amyloid fibril formation, aggregation ordeposition. The agents of the invention may act to, ameliorate thecourse of fibril formation; inhibiting neurodegeneration or cellulartoxicity induced by amyloid-β; inhibiting amyloid-β inducedinflammation; enhancing the clearance of amyloid-β from the brain; orfavoring greater catabolism of Aβ.

An agent may be effective in controlling amyloid-β deposition by actingdirectly on brain Aβ, e.g., by maintaining it in a non-fibrillar form orfavoring its clearance from the brain. The compounds may slow down APPprocessing; may increase degradation of Aβ fibrils by macrophages or byneuronal cells; or may decrease Aβ production by activated microglia.These agents could also prevent Aβ in the brain from interacting withthe cell surface and therefore prevent neurotoxicity, neurodegeneration,or inflammation.

An agent identified by a method provided herein may be used to treatAlzheimer's disease (e.g., sporadic or familial AD). The agent may alsobe used prophylactically or therapeutically to treat other clinicaloccurrences of amyloid-β deposition, such as in Down's syndromeindividuals and in patients with cerebral amyloid angiopathy (“CAA”),hereditary cerebral hemorrhage, or early Alzheimer's disease.

The agent may be used to treat mild cognitive impairment. Mild CognitiveImpairment (“MCI”) is a condition characterized by a state of mild butmeasurable impairment in thinking skills, which is not necessarilyassociated with the presence of dementia. MCI frequently, but notnecessarily, precedes Alzheimer's disease.

Additionally, abnormal accumulation of APP and of amyloid-β protein inmuscle fibers has been implicated in the pathology of sporadic inclusionbody myositis (IBM) (Askanas, V., et al. (1996) Proc. Natl. Acad. Sci.USA 93: 1314-1319; Askanas, V. et al. (1995) Current Opinion inRheumatology 7: 486-496). Accordingly, agents identified by a methodprovided herein amy be used prophylactically or therapeutically in thetreatment of disorders in which amyloid-β protein is abnormallydeposited at non-neurological locations, such as treatment of EBM bydelivery of the compounds to muscle fibers.

Additionally, it has been shown that Aβ is associated with abnormalextracellular deposits, known as drusen, that accumulate along the basalsurface of the retinal pigmented epithelium in individuals withage-related macular degeneration (ARMD). ARMD is a cause of irreversiblevision loss in older individuals. It is believed that Aβ depositioncould be an important component of the local inflammatory events thatcontribute to atrophy of the retinal pigmented epithelium, drusenbiogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc. Natl.Acad. Sci. USA 99(18), 11830-5 (2002)).

Accordingly, the invention relates generally to methods of treating orpreventing an amyloid-related disease in a subject (preferably a human)comprising administering to the subject a therapeutic amount of an agentor compound identified by a method provided herein, such that amyloidfibril formation or deposition, neurodegeneration, or cellular toxicityis reduced or inhibited. In another embodiment, the invention relates toa method of treating or preventing an amyloid-related disease in asubject (preferably a human) comprising administering to the subject atherapeutic amount of a compound identified by a method describedherein, such that cognitive function is improved or stabilized orfurther deterioration in cognitive function is prevented, slowed, orstopped in patients with brain amyloidosis, e.g., Alzheimer's disease,Down's syndrome or cerebral amyloid angiopathy. These compounds can alsoimprove quality of daily living in these subjects.

Further, the present invention relates to pharmaceutical compositionscomprising agents for the treatment of an amyloid-related disease, aswell as methods of manufacturing such pharmaceutical compositions.

In general, the agents identified by methods provided herein may beprepared by any method known to the skilled artisan. The agents of theinvention may be supplied in a solution with an appropriate solvent orin a solvent-free form (e.g., lyophilized). In another aspect of theinvention, the agents and buffers necessary for carrying out the methodsof the invention may be packaged as a kit. The kit may be commerciallyused according to the methods described herein and may includeinstructions for use in a method of the invention. Additional kitcomponents may include acids, bases, buffering agents, inorganic salts,solvents, antioxidants, preservatives, or metal chelators. Theadditional kit components are present as pure compositions, or asaqueous or organic solutions that incorporate one or more additional kitcomponents. Any or all of the kit components optionally further comprisebuffers.

The therapeutic agent may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

To administer the therapeutic agent by other than parenteraladministration, it may be necessary to coat the agent with, orco-administer the agent with, a material to prevent its inactivation.For example, the therapeutic agent may be administered to a subject inan appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., J. Neuroimmunol. 7, 27(1984)).

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

Suitable pharmaceutically acceptable vehicles include, withoutlimitation, any non-immunogenic pharmaceutical adjuvants suitable fororal, parenteral, nasal, mucosal, transdermal, intravascular (IV),intraarterial (IA), intramuscular (IM), and subcutaneous (SC)administration routes, such as phosphate buffer saline (PBS).

The vehicle can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents are included, for example, sugars, sodiumchloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic agent in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic agent into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and freeze-drying which yields a powder of the activeingredient (i.e., the therapeutic agent) plus any additional desiredingredient from a previously sterile-filtered solution thereof.

The therapeutic agent can be orally administered, for example, with aninert diluent or an assimilable edible carrier. The therapeutic agentand other ingredients may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe subject's diet. For oral therapeutic administration, the therapeuticagent may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic agent in the compositions and preparations may, of course,be varied. The amount of the therapeutic agent in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic agent calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical vehicle. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the therapeutic agent and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such a therapeutic agent for the treatment of amyloiddeposition in subjects.

The present invention therefore includes pharmaceutical formulationscomprising agents identified by methods described herein, includingpharmaceutically acceptable salts thereof, in pharmaceuticallyacceptable vehicles for aerosol, oral and parenteral administration.Also, the present invention includes such agents, or salts thereof,which have been lyophilized and which may be reconstituted to formpharmaceutically acceptable formulations for administration, as byintravenous, intramuscular, or subcutaneous injection. Administrationmay also be intradermal or transdermal.

In accordance with the present invention, an agent, and pharmaceuticallyacceptable salts thereof, may be administered orally or throughinhalation as a solid, or may be administered intramuscularly orintravenously as a solution, suspension or emulsion. Alternatively, theagents or salts may also be administered by inhalation, intravenously orintramuscularly as a liposomal suspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired agent, or a salt thereof, or aplurality of solid particles of the agent or salt. The desiredformulation may be placed in a small chamber and nebulized. Nebulizationmay be accomplished by compressed air or by ultrasonic energy to form aplurality of liquid droplets or solid particles comprising the agents orsalts. The liquid droplets or solid particles should have a particlesize in the range of about 0.5 to about 5 microns. The solid particlescan be obtained by processing the solid agent, or a salt thereof, in anyappropriate manner known in the art, such as by micronization. The sizeof the solid particles or droplets will be, for example, from about 1 toabout 2 microns. In this respect, commercial nebulizers are available toachieve this purpose.

A pharmaceutical formulation suitable for administration as an aerosolmay be in the form of a liquid, the formulation will comprise awater-soluble agent, or a salt thereof, in a carrier which compriseswater. A surfactant may be present which lowers the surface tension ofthe formulation sufficiently to result in the formation of dropletswithin the desired size range when subjected to nebulization.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically acceptable vehiclessuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, tragacanth, and sodium alginate; typical-wetting agentsinclude lecithin and polysorbate 80; and typical preservatives includemethyl paraben and sodium benzoate. Peroral liquid compositions may alsocontain one or more components such as sweeteners, flavoring agents andcolorants disclosed above.

Pharmaceutical compositions may also be coated by conventional methods,typically with pH or time-dependent coatings, such that the subjectagent is released in the gastrointestinal tract in the vicinity of thedesired topical application, or at various times to extend the desiredaction. Such dosage forms typically include, but are not limited to, oneor more of cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, andshellac.

Other compositions useful for attaining systemic delivery of the subjectagents include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypiopylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

The compositions of this invention can also be administered topically toa subject, e.g., by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject, or transdermallyvia a “patch”. Such compositions include, for example, lotions, creams,solutions, gels and solids. These topical compositions may comprise aneffective amount, usually at least about 0.1%, or even from about 1% toabout 5%, of an agent of the invention. Suitable carriers for topicaladministration typically remain in place on the skin as a continuousfilm, and resist being removed by perspiration or immersion in water.Generally, the carrier is organic in nature and capable of havingdispersed or dissolved therein the therapeutic agent. The carrier mayinclude pharmaceutically acceptable emolients, emulsifiers, thickeningagents, solvents and the like.

The working examples provided below are to illustrate, not limit, thedisclosure. Various parameters of the scientific methods employed inthese examples are described in detail below and provide guidance forpracticing the disclosure in general.

EXAMPLES Example 1

cDNAs for G-proteins Gα_(oA) and Gα_(oB) in pcDNA3 were purchased fromUMR cDNA Resource Center, Rolla, Mo. Full-length human PS-1 and PS-2cDNAs in pcDNA3 were cloned by PCR as already described. Tail-lessconstructs of PS-1 and PS-2 were constructed in pcDNA3 in which only thecytoplasmic domain of PS-1 or PS-2 immediately following the lastTM-domain is deleted (this construct comprises of amino acids 1-430 ofPS-1 and 1-410 of PS-2).

Cell culture: ES (PS-1^(−/−)/PS-2^(−/−)) cells were cultured accordingto published protocols.

Transfections: ES (PS-1^(−/−)/PS-2^(−/−)) were transiently transfectedwith 15 μg of pcDNA constructs of full-length human PS-1 or PS-2 and thecDNA of the desired G-proteins using the lipofectamine (Invitrogen)method. Briefly, the lipofectamine—DNA solution was be left at roomtemperature for 30 mins, mixed with enough serum-free medium and addedto the cells. Cells were incubated for 5 h at 37° C. in a CO₂ incubatorafter which the medium was replenished with serum and cells harvested12-24 hours after transfection.

Immunoprecipitations: 24 h after transfection, the culture medium wasremoved, and cells scraped in 200 μl of extraction buffer. Wholecell-extracts were made by sonication, using the solubilizationconditions of Smine et al (50 mM HEPES/NaOH, pH 7.4, 1 mM EDTA, 1 mMDTT, 1% Triton X-100, 60 mM octylglycoside and protease inhibitors). 100μg of each extract was immunoprecipitated using monoclonal antibodies tothe large loop of PS-1 (MAB5232) or PS-2 (MA1-754). Theimmunoprecipitated proteins were next separated on 12% SDS PAGE andtransferred to a membrane. Western blot hybridization against antibodiesto the G protein G_(o) (K-20, sc-387 from Santa Cruz Biotechnology,affinity purified; this polyclonal antibody recognizes both G_(oA) andG_(oB)) was then carried out.

Western blot hybridizations: Immunoprecipitated proteins were boiled for5 min in loading buffer (50 mM Tris, pH 6.8, 0.1 M DTT, 2% SDS, 0.1%bromophenol blue, 10% glycerol), separated electrophoretically onSDS-PAGE (12%) gels, and the proteins transferred onto nitrocellulosefilters. Filters were incubated with the primary polyclonal rabbitG-protein antibodies followed by horse radish peroxidase-conjugated goatanti-rabbit IgG. Filter-bound peroxidase activity was detected bychemiluminescence.

Binding of G-protein G_(o) to PS-1 ES (PS-1^(−/−)/PS-2^(−/−)) cells weretransiently transfected with cDNA to full-length human PS-1 and the cDNAof the G-proteins G_(oA) or G_(oB) (UMR cDNA Resource Center, Rolla,Mo.). 24 h after transfection, whole cell-extracts were made bysonication, using the solubilization conditions of 5 mine et al (50 mMHEPES/NaOH, pH 7.4, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 60 mMoctylglycoside and protease inhibitors). 100 μg of each extract wasimmunoprecipitated using monoclonal antibodies to the large loop, whichis extracellular in the 7-TM model (Mab # 5232, Chemicon, which was usedin previous published work). The immunoprecipitated proteins were nextseparated on 12% SDS PAGE and transferred to a membrane. Western blothybridization against antibodies to both, PS-1 and G_(o) (K-20, sc-387from Santa Cruz Biotechnology, affinity purified; this polyclonalantibody recognizes both G_(oA) and G_(oB)) was then carried out.

Binding of G-protein G_(o) to PS-2: ES (PS-1^(−/−)/PS-2^(−/−)) weretransiently transfected with cDNA of full-length human PS-2 and the cDNAof the G-proteins G_(oA) or G_(oB) (UMR cDNA Resource Center, Rolla,Mo.). 24 h after transfection, whole cell-extracts were made bysonication, using the solubilization conditions of 5 mine et al (50 mMHEPES/NaOH, pH 7.4, 1 mM EDTA, 1 mM DTT, 1% Triton X-100, 60 mMoctylglycoside and protease inhibitors). 100 μg of each extract wasimmunoprecipitated using mouse monoclonal antibodies to the large loopof PS-2 (MA1-754 from Affinity BioReagents). The immunoprecipitatedproteins were next separated on 12% SDS PAGE and transferred to amembrane. Western blot hybridization against antibodies to both, PS-2and G_(o) was then carried out.

Pertussis Toxin Treatment: The PTx protomer was incubated with 10 mM DTTat 37° C. for 10 min to convert it to its enzymatically active form. 5 hafter transfecting ES cells with PS-1 or PS-2 and the G-protein cDNAs,500 ng/ml of activated PTx was added to the cells in culture medium inthe presence of 1 mM NAD, 2 mM MgCl₂ and 1 mM EDTA and the cellsincubated at 37° C. in the presence of 5% CO₂ for 12 h. Cells were thenharvested and examined for [³⁵S]GTPγS incorporation as described below.

GTPγS Binding: Cells were harvested and proteins solubilized bysonication in solublilization buffer (50 mM HEPES/NaOH pH 7.4, 1 mMEDTA, 1 mM DTT, 1% Triton-X100, 60 mM octylglycoside, 1× Proteaseinhibitor mix). 100 μg of protein was mixed with an equal volume ofBuffer B (50 mM HEPES/NaOH pH 7.4, 40 μM GDP, 50 mM MgCl₂, 100 mM NaCl)in a volume of 200 μl. The reaction was started with 50 nM [³⁵S]GTPγS(1250 Ci/mmol) and incubation carried out for 60 min at RT after whichthe reaction was stopped by the addition of 20 μl of 10× Stopping buffer(100 mM Tris-Hcl, pH 8, 25 mM MgCl₂, 100 mM NaCl, 20 mM GTP. The samplewas then immunoprecipitated with anti-PS-1 loop monoclonal antibody (5μl). The antibody-protein complex was subjected to binding to ProteinA/G agarose for 90 min at RT and washed twice with washing buffer 1 (50mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 1% Triton X-100 1X proteaseinhibitor mix, 150 mM NaCl and 60 mM octyl-β-D-glucopyranoside), andonce with each of washing buffers 2 (50 mM HEPES, pH 7.4, 1 mM EDTA, pH8.0, 0.5% Triton X-100, 1× protease inhibitor mix and 50 mM NaCl) and 3(50 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0 and 1× protease inhibitor mix.The washed agarose beads were then suspended in scintillation fluid(CytoScint, ICN) (5 ml) and counted in a Beckman Coulter LS 6000 SCscintillation counter for 3 min.

When 100 μg of extract of ES (PS-1^(−/−)/PS-2^(−/−)) cellsco-transfected with cDNAs for full-length human PS-1 and the G-proteinGα_(OA) or Gα_(oB) were immunoprecipitated with MAb to the largehydrophilic loop of PS-1, followed by Western blot hybridization withaffinity purified polyclonal antibody to G_(o) (which recognizes bothisoforms, G_(oA) and G_(oB)), only the PS-1/G_(oA) co-transfected cellsgave a robust signal for G_(o) at ˜45 kDa (FIG. 1, lane 3), suggestingthat GOA, but not GOB, binds to PS-1. Control untransfected cells orcells transfected with PS-1 alone did not show a G_(o) band on Westernblots when treated identically (FIG. 1).

Verification of the binding of G-protein G_(o) to the cytoplasmiccarboxyl terminus of PS-1. A tail-less construct of PS-1 was made inpcDNA3 in which only the cytoplasmic domain of PS-1 immediatelyfollowing the last TM-domain is deleted (this construct comprises aminoacids 1-430). This construct was used to transfect ES(PS-1^(−/−)/PS-2^(−/−)) cells. Tail-less PS-1 has been shown tointegrate into the membrane and to be expressed at the cell surface. Inan identical strategy to the one described above for full-length PS-1,ES (PS-1^(−/−)/PS-2^(−/−)) cells were transfected with cDNAs fortail-less PS-1 and the G-proteins G_(oA) or G_(oB). Cells extracts werethen subjected to immunoprecipitation with PS-1 loop MAb # 5232),separated on SDS PAGE and Western blotted with antibodies to G_(o).

100 μg of extract of ES (PS-1^(−/−)/PS-2^(−/−)) cells co-transfectedwith cDNAs for tail-less PS-1 and the G-protein Gα_(oA) or Gα_(oB) wereimmunoprecipitated with MAb to the large hydrophilic loop of PS-1,followed by Western blot hybridization with affinity purified polyclonalantibody to G_(o) (recognizes both isoforms, G_(oA) and G_(oB)). Bindingwas detected (FIG. 1, lane 6) indicating that the carboxyl terminal 39amino acids earlier identified to be the binding domain did notconstitute the entire binding domain of PS-1 for G_(oA). G_(oB) showedno binding to tail-less PS-1 (FIG. 1, lane 7).

The results using the tail-less construct, which eliminated the majorpart of G_(oA) binding to PS-1, show specificity for some PS-1:G_(oA)binding to another region of PS-1 besides the PS-1 tail. They also ruleout the possibility that G_(oA) may have bound to other components ofthe PS-1 β-secretase complex, that may have co-immunoprecipitated withthe PS-1 antibody.

Additional studies were performed to elucidate the binding of G-proteinG_(o) to intact PS-2. The 39 amino acid PS-1 C-terminal regionidentified to be the binding domain is completely conserved in theC-terminal tail of PS-2. Accordingly, it was believed that theC-terminal domain of PS-2 would also bind Gα_(o). As with PS-1, G_(o)was shown to bind to PS-2, but with distinct differences. The G_(o)antibody, which recognizes both G_(oA) and G_(oB), showed a doublet onWestern blots of PS-2 immunoprecipitates of extracts of cellsco-transfected with PS-2 and G_(oA) as well as PS-2 and G_(oB) cDNAs.The doublet presumably represents binding of both isoforms of G_(o) toPS-2 (FIG. 3, lanes 2 and 4). In contrast, PS-1 did not bind to G_(oB)(FIG. 1, lane 4) and only showed a single band on Western blots with thesame G_(o) antibody (FIG. 1, lane 3).

The binding of G-protein G_(o) to the cytoplasmic carboxyl terminus ofPS-2 was investigated. As for PS-1, a tail-less construct of PS-2 wasmade in pcDNA3 in which only the cytoplasmic domain of PS-2 immediatelyfollowing the last TM-domain was deleted (this construct comprised aminoacids amino acids 1-410). This construct was used to transfect ES(PS-1^(−/−)/PS-2^(−/−)) cells and has been shown to integrate into themembrane and be expressed at the cell surface (FIG. 2). In an identicalstrategy to the one described above for full-length PS-1 and PS-2, ES(PS-1^(−/−)/PS-2^(−/−)) cells were transfected with cDNAs for tail-lessPS-2 and the G-proteins G_(oA) and G_(oB). Cells extracts were thensubjected to immunoprecipitation with PS-2 loop Mab # MA1-754),separated on SDS PAGE and Western blotted with antibodies to G_(o).

When tail-less PS-2, co-expressed with G_(oA) was immunoprecipitatedwith PS-2 MAb and Western blotted with anti G_(o) antibody, as withresults for PS-1, there was a decrease in band intensity, but the bandwas not totally absent. The intensity of the bands in the G_(oB)/PS-2co-transfection sample, on the other hand, was unaltered for thetail-less sample suggesting that G_(oB) binds PS-2 at an intracellulardomain other than the carboxyl terminal tail FIG. 3, lanes 3 and 5).Therefore, PS-1 and PS-2 are discriminated not only by the G_(o)isoforms that they bind to, but also the binding sites on the PS-1 andPS-2 that are not homologous to one another. It seems likely, therefore,that functional studies of PS-1 and PS-2 will give quite differentresults; i.e., PS-1 and PS-2 are not merely functionally redundantproteins.

Additional studies of PS mediated functional activation of Gα_(oA) andGα_(oB) PS-1 and the G-proteins G_(oA) and G_(oB) were performed.Previous studies used GTP hydrolysis and GTPγS binding as one of severalindependent approaches to evaluate G_(o) binding to the carboxylterminus of PS-1. However, they carried out this assay with asynthesized peptide of residues 429-467 in the C-terminus of PS-1, alongwith three control peptides. The approach on the other hand was toevaluate the functional consequences of the binding of the G-proteinsG_(oA) and G_(oB) to intact PS-1 and PS-2 in the co-transfected cell, byassaying for ³⁵S-GTPγS incorporation in cell extracts.

The ³⁵S-GTPγS incorporation in extracts of ES cells that wereco-transfected with cDNAs for PS-1 and the G-protein G_(oA) was shown tobe over 700% the value obtained for control untransfected ES (PS^(−/−))cells (FIG. 4, lane 2). This increase was not seen when cellstransfected with PS-1 and G_(oA) cDNAs were first treated with PTx (FIG.4, lane 3) showing an inhibition of function in the presence of thetoxin. Cells transfected with cDNAs for PS-1 and G_(oB) on the otherhand did not show incorporation of ³⁵S-GTPγS (FIG. 4 lane 4), consistentwith previous results of a lack of binding of G_(oB) to PS-1.

As with PS-1, PS-2 when co-expressed with G_(oA) and assayed for³⁵S-GTPγS binding showed greater than 700% increase in ³⁵S-GTPγS bindingover untransfected control ES (PS^(−/−)) extracts (FIG. 4, lane 2). Thiswas inhibited in the presence of PTx (FIG. 4, lane 3). Unlike the casefor PS-1, GOB binding to PS-2 does give an increase in ³⁵S-GTPγSincorporation. This novel finding is consistent with other data providedherein indicating that G_(oB) binds to PS-2 but not PS-1. The increasein ³⁵S-GTPγS incorporation is less than that observed for G_(oA) (˜300%)(FIG. 4, lane 4). This increase is inhibited in the presence of PTx. Theresults shown in FIG. 4 are representative of at least 3 independentexperiments.

Example 2

ES PS double-null cells were cultured and plated overnight. The cellswere transfected with a pcDNA3 construct of full-length human β-APP cDNAusing lipofectamine (Invitrogen) according to the manufacturer'sprotocols. DAMI cells were cultured and transfected either with pcDNA3or with a pcDNA3 construct of full-length human PS-1 or PS-2 cDNA.

Affinity-purified polyclonal rabbit anti-PTyr antibodies (Maher et al.,1985) were used in Western blots and were a kind gift from Dr. ElenaPasquale. A mouse monoclonal anti-PTyr antibody (4G10; UpstateBiotechnology, Lake Placid, N.Y.) was used in ELISA analyses. Mousemonoclonal antibody to human pp60c-src (Anti-Src, clone GD11) and rabbitpolyclonal antibody to Lyn (Anti-Lyn) were purchased from UpstateBiotechnology. Rabbit polyclonal antibody to Fyn (Anti-Fyn, sc-16) waspurchased from Santa Cruz Biotechnology, Santa Cruz, Calif. Primary ratanti-human PS-1 monoclonal antibody MAb #1563 directed to the N-terminaldomain of PS-1 was purchased from Chemicon International, Temecula,Calif. It was raised to a fusion protein antigen containing part of theN-terminal domain of human PS-1 (residues 21-80) fused to GST. Primarymouse monoclonal antibody MAb #348 to the human β-APP extracellulardomain was purchased from Chemicon International.

Fluorescein isothiocyanate (FITC)-conjugated affinity purified goatanti-rat IgG and tetramethylrhodamine β isothiocyanate(TRITC)-conjugated affinity purified donkey anti-mouse IgG secondaryantibodies were purchased from Jackson ImmunoResearch, West Grove, Pa.Immunofluorescence labeling Transfected and untransfected DAMI cellswere fixed with 4% paraformaldehyde in PBS for 10 mins and used withoutpermeabilization. Cells were labeled in suspension with antisera to PS-1(1:200 dilution), and β-APP (1:500 dilution) in PBS containing 1% BSAfor 30 min at room temperature. After washing with PBS three times bycentrifugation, the cells were resuspended in 1% BSA/PBS and incubatedwith appropriate fluorescent secondary antibodies. Incubation wascarried out at room temperature for 20 min, after which the cells werewashed with PBS and mounted onto slides in the presence of mountingmedium (Vector Laboratories, Burlingame, Calif.).

Immunofluorescent microscopy was performed using oil immersion with aX60 objective lens. The slides were viewed using fluoresceinisothiocyanate and tetramethylrhodamine β isothiocyanate filters and aZeiss Photoscope III instrument, or with Nomarski optics.

N-terminal domains of PS-1 and PS-2 were obtained by PCR and cloned intothe Tth 111 I and Xho-1 sites of the FLAG expression vector (ScientificImaging Systems, IBI 13100) to produce a fusion protein with FLAGattached at the N-terminus of either the PS-1- or 2 N-terminal domains.The two FLAG-fusion proteins were grown separately in DH5α bacteria andaffinity purified according to the manufacturer's protocols. Thepurified recombinant proteins were checked by Western blots usingantibodies to both FLAG and either the N-terminal domain of PS-1 orPS-2.

DAMI:ES cells: Equal numbers (0.5×10⁶/ml) of β-APP 695 (Selkoe andPodlisny, 2002)-transfected ES double-null cells and PS-1 transfectedDAMI cells were co-cultured at 37° C. for various times between 0-20mins.

All experiments after those in FIG. 7 (with the exception of FIG. 9 a,Panel 4) were carried out with appropriately transfected DAMI cellsonly. Equal numbers (0.5×10⁶/ml) of β-APP-transfected DAMI cells andeither PS-1- or PS-2-transfected DAMI cells were mixed gently at roomtemperature, exactly as described (Dewji and Singer, 1998). In controlexperiments, DAMI cells transfected with pcDNA3 alone were substitutedfor the β-APP transfected cells.

At several times between 0 and 20 min after mixing, an aliquot of eachcell mixture was rapidly centrifuged, the culture medium was removed,and the cell pellet was suspended in 200 μl of extraction buffer (50 mMTris, pH 8.0/150 mM NaCl/0.5% Nonidet-P40) containing proteaseinhibitors (1 mM 4-(2-aminoethyl) benzene sulfonyl fluoridehydrochloride (AEBSF)/1 μg/ml antipain/0.1 μg/ml pepstatin A/0.1 μg/mlleupeptin) and the phosphatase inhibitor sodium orthovanadate (0.1 mM).The mixture was sonicated with three bursts of 20 sec duration and thencentrifuged. These extract supernatants were then used for Western blotand ELISA analyses as described below.

Assays for Src family of protein tyrosine kinases in cell extracts wereperformed. The substrate peptide {[Lys19] cdc2 (6-20)-NH2} and controlpeptides {[Lys19Ser14Val12]cdc2 (6-20)} and {[Lys19Phe15]cdc2(6-20)}were purchased from Upstate Biotechnology Inc. Src kinase activity wasmeasured in extracts of transfected DAMI cells (either β-APP- orpcDNA3-transfected) mixed with PS-1-transfected cells; and with β-APP-or pcDNA3-transfected cells mixed with PS-2-transfected cells, using allthree peptides. Controls included experiments carried out using nosubstrate in the reaction mixture.

The substrate peptide (1.5 mM in 10 μl), Src kinase reaction buffer (100mM Tris-HCl, pH 7.2, 125 mM MgCl₂, 25 mM MnCl₂, 2 mM EGTA, 0.25 mMsodium orthovanadate, 2 mM DTT) (10 μl), Src kinase (2-20 U of purifiedenzyme per assay or 10-200 μg protein lysate in 10 μl and [γ-³²P]ATP(NEN Dupont, Boston, Mass.) diluted with Mn 2+/ATP cocktail (10 μl),were incubated for 15-20 min at 30° C.

Aliquots of the extract supernatants described above (100 μgprotein/lane) were boiled for 5 min in loading buffer (50 mM Tris, pH6.8, 0.1 M DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), separatedelectrophoretically on SDS-PAGE (10%) gels, and the proteins transferredonto nitrocellulose filters. Filters were incubated with the primarypolyclonal rabbit anti-PTyr antibodies followed by the horse radishperoxidase-conjugated goat anti-rabbit IgG. Filter-bound peroxidaseactivity was detected by chemiluminescence.

Cell lysates were prepared in extraction buffer and clarified bymicro-centrifugation at 4° C. for 15 mins.

Extracts were incubated with 4 μg antibodies specific for either c-Src,Lyn or Fyn followed by protein-A or G sepharose (40 μl of slurry). Theantigen antibody-protein-A (or -G) sepharose complex was washed threetimes in RIPA (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1%Na deoxycholate, 0.1% SDS, 1% trasylol, 25 μM leupeptin) containing 300mM NaCl, once with RIPA containing 10 mM NaCl, twice with 40 mMTris-HCl, pH 7.2 and once with kinase buffer containing 25 mM HEPES, pH6.9, 3 mM MnCl₂ and 200 μM sodium orthovanadate.

Reactions were performed according to published protocols (Zisch et al.,1998) in 40 μl kinase buffer (25 mM Hepes, pH 6.9, 3 mM MnCl₂ and 200 μMsodium orthovanadate) containing 5 μCi [g32P] ATP (3000 Ci/mmol) for 30min at 37° C. The reaction beads were washed three times with kinasebuffer and resuspended in 75 μl SDS gel loading buffer (250 mM Tris-HCl,pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 0.02% bromophenol blue and 75%glycerol). Autophosphorylation reactions were subjected to SDS-PAGEfollowed by transfer of proteins onto PVDF membranes andautoradiography.

ELISAs Protein tyrosine kinase activity was measured by an Enzyme LinkedImmunosorbent Assay (ELISA) using a tyrosine kinase assay kit (UpstateBiotechnology). A biotinylated substrate peptide containing tandemrepeats of Poly (Glu4-Tyr) was incubated with supernatants of extractsof transfected cells mixed for different times (20 μg protein/well) inthe presence of non-radioactive ATP and a Mn²⁺/Mg²⁺ co-factor cocktailaccording to the manufacturer's protocols. A phosphotyrosine specificmouse monoclonal antibody (4G10) conjugated to horseradish peroxidasewas used to detect the phosphorylated substrate by ELISA.

Absence of cell surface expression of β-APP in untransfected andPS-1-transfected DAMI cells. Because the initial set of studies dependson the proposition that DAMI cells, after transfection with PS-1,continue to express only negligible amounts of β-APP on their surface,the following experiments were first carried out. Both untransfected andPS-1-transfected DAMI cells in the fixed but impermeable state weredoubly immunofluorescently labeled for β-APP and PS-1. Untransfectedfixed impermeable DAMI cells, as previously shown (Querfurth and Selkoe,1994), do not express significant amounts of β-APP at the cell surface(FIG. 5 a, Panel 2), whereas DAMI cells transfected with a pcDNA3construct of β-APP show substantial cell-surface expression in fixedimpermeable cells (FIG. 5 b, Panel 2). FIGS. 5 a and b, Panels 1 show,however, that untransfected fixed impermeable DAMI cells do expressendogenous cell-surface PS-1. In FIG. 5 c, Panel 1, this cell-surfaceexpression of PS-1 is increased in fixed impermeable PS-1-transfectedcells. FIG. 5 c, Panel 2, shows that transfecting DAMI cells with PS-1does not significantly increase the cell-surface expression of β-APPover the negligible levels seen in untransfected cells (FIG. 5 a, Panel2). FIG. 5 d, Panel 2, shows cell-surface expression of β-APP in ESdouble-null fixed impermeable cells transfected with β-APP, but not PS-1expression (FIG. 5 d, Panel 1).

With untransfected, fixed impermeable ES double-null cells, there is, asexpected, no labeling for cell-surface PS-1 (FIG. 5 e, Panel 1), but asmall amount of surface expression of endogenous β-APP (FIG. 5 e, Panel2). These results confirm that in interactions of β-APP-transfected ESdouble-null cells and PS-transfected DAMI cells, only the ES cellsexpress cell-surface β-APP, and no PS; while only the PS-transfectedDAMI cells express PS, and no β-APP, at the cell-surface. If a β-APP:PSinteraction occurs after cell mixing, it can therefore only be theresult of a cell-cell interaction.

Also provided herein are data indicating that specific β-APP:PSintercellular signaling results in an increase in tyrosine kinaseactivity. ES double-null cells transfected with β-APP were mixed withDAMI cells transfected with PS-1, and were co-cultured for various timesbetween 0-20 min, using cell densities that ensured cell-cell contact.ELISA assays were then carried out on cell extracts to measure proteintyrosine kinase activity. FIG. 6 line “a” shows that these co-culturesproduced a rapid and transient increase in protein tyrosine kinaseactivity similar in extent and kinetics to those previously describedwhen PS-1-transfected DAMI cells were mixed with β-APP-transfected DAMIcells (Dewji and Singer, 1998). When the same interaction as in FIG. 6line “a” was carried out in the presence of 25 μg of purifiedbaculovirus-derived soluble β-APP (extra-cellular domain of β-APP) (FIG.6 line “b”) or 25 μg of fusion peptide of the FLAG reporter fused to theN-terminal domain of PS-1 (FIG. 6 line “c”), no increase in proteintyrosine kinase activity resulted. On the other hand, the sameβ-APP:PS-1 co-cultures in the presence 25 μg of FLAG-PS-2 N-terminaldomain fusion peptide did not inhibit PTyr formation (FIG. 6 line “d”).These results clearly establish several points: 1) Soluble β-APP itselfdoes not activate the PS-1-transfected DAMI cells to exhibit tyrosinekinase activity; the intact β-APP in the transfected ES cell membrane isrequired. On the contrary, the soluble β-APP inhibits the activityproduced by the membrane-bound β-APP, demonstrating that membrane-boundβ-APP is specifically involved in the activation; 2) the N-terminaldomain of PS-1 is itself incapable of activating the β-APP-transfectedcell to exhibit tyrosine kinase activity. The intact PS-1 molecule inits DAMI cell membrane is required. But the N-terminal domain of PS-1(but not PS-2) inhibits the activation of the co-culture, showing thatmembrane-bound PS-1 on the PS-1-transfected DAMI cell is alsospecifically involved in the interaction; 3) The protein nature of theinhibitors, soluble β-APP and the FLAG-fusion protein of the N-terminaldomain of PS-1, assures their impenetrability of the cell membranes ofliving DAMI and ES cells, and therefore demonstrates that it is only theexterior domains of the cell-surface β-APP and PS-1 that are involved ingenerating the signaling event (i.e., the signaling is of the juxtacrinetype). These results provide compelling evidence that establish that ajuxtacrine interaction between β-APP and PS can occur.

Furthermore, this demonstration that the N-terminal domain of PS-1 isexposed at the extracellular surface is consistent with the 7-TMtopography of the PS proteins, but is contrary to the prediction of the8-TM model, which positions the N-terminal domain of PSintra-cellularly.

Additional data provided herein indicate that β-APP:PS-1 and β-APP:PS-2intercellular signaling can be mediated by members of the Src family oftyrosine kinases. The increases in PTyr modification that are aconsequence of β-APP:PS intercellular binding involved one or moreprotein tyrosine kinases that need to be identified. Since neither β-APPnor the PS proteins contain such a kinase active site, an indirectactivity of the cytoplasmic domains of these proteins, such as thedirect or indirect binding of a cytoplasmic tyrosine kinase to one ofthese domains, may be involved in the downstream signal. Since severalcytoplasmic tyrosine kinases have been identified within the Src genefamily, Src family protein tyrosine kinases were assayed in cellextracts of mixed transfected cells using the substrate peptide[lys19]cdc2(6-20)-NH₂ (KVEKIGTYGVVKK (SEQ ID NO:4)). This peptide, withTyr 19 in cdc2(6-20) replaced by lys, has been shown to be an efficientsubstrate for the Src family kinases. All Src family kinases tested,including v-Src and c-Src, c-Yes, Lck, Lyn and Fyn, demonstrate strongactivity towards this substrate. Two control peptides were also used: Inthe first peptide, [lys19ser14val12]cdc2(6-20)NH2 (KVEKIGVGSYGWKK (SEQID NO:5)), glu 12 and thr 14 were replaced by val and ser, respectively,causing a significant decrease in efficiency of the resulting peptide toserve as a substrate for the Src family tyrosine kinases. The otherpeptide, [lys19phe15]cdc2(6-20)NH2 (KVEKIGEGTFGWKK (SEQ ID NO:6)) shouldnot be phosphorylated by tyrosine kinases but did contain a potentialtarget for ser/thr kinases (thr 14).

The results of Src family kinase activity measurements in extracts ofco-cultures of β-APP-transfected DAMI cells with PS-1-transfected DAMIcells evoking β-APP:PS-1 interactions, and for the corresponding controllacking β-APP (pcDNA3:PS-1), are shown in FIGS. 7 a and b. Similarresults for transfected DAMI cell mixtures evoking β-APP:PS-2interactions, and extracts of control pcDNA3:PS-2 mixed transfected DAMIcells, using these three peptides are shown in FIGS. 7 c and d. For eachβ-APP:PS cell mixture, where [lys19]cdc2(6-20)NH2 was u sed as the Srcfamily kinase substrate, the temporal course of increased activitycompared to control peptides were obtained that paralleled ELISA resultsfor tyrosine kinase activity. For the β-APP:PS-1 interaction (FIG. 7 a),Src family kinase activity peaked at 8 minutes and returned to baselinelevels by 12 minutes confirming previous ELISA results for tyrosinekinase activity as a function of time after cell mixing. No significantincrease could be observed when the same substrate was used for thecontrol pcDNA3:PS-1 (FIG. 7 b) mixed cells. For the cell mixturesevoking β-APP:PS-2 interactions (FIG. 7 c), as for the tyrosine kinaseELISA results, two clear peaks of activity were observed with substratepeptide [lys19]cdc2(6-20)NH₂, at 9 and 16 minutes after mixing.

For the corresponding control lacking β-APP, pcDNA3:PS-2 (FIG. 7 d), nosignificant increases of Src kinase activity over background wereobserved. These results suggest that the increases in tyrosine kinaseactivity previously observed for β-APP—with PS-1-transfected cellmixtures, or P-APP—with PS-2-transfected cell mixtures, involve one ormore members of the Src tyrosine kinase family.

Inhibition of tyrosine kinase activity in the presence of specificinhibitors of Src family kinases and tyrosine kinase. The involvement ofthe Src kinase family in β-APP:PS intercellular signaling was furtherconfirmed with ELISAs of extracts of β-APP:PS-1 mixed cell interactionscarried out in the presence or absence of specific inhibitors oftyrosine kinase (herbimycin A) and Src family kinases (PP2). FIG. 8 ademonstrates that in the presence of 10 μg/ml herbimycin A, the increasein tyrosine kinase activity at 8-10 mins after mixing β-APP-transfectedDAMI cells with PS-1-transfected DAMI cells is completely inhibited. Thesame experiment carried out in the presence of 10 nM PP2 (FIG. 8 b)similarly showed the inhibition of tyrosine kinase activity.

Additional data related to the involvement of c-Src in β-APP:PS-1intercellular signaling is provided below. In order to determine theidentity of the Src family member(s) involved in the β-APP:PS-1intercellular signaling, we began by investigating pp60c-Src. Two mainprotein bands of apparent molecular weights 58 and 60 kDa, a doubletsimilar in size to c-Src, underwent transient PTyr modification in thisjuxtacrine interaction. When extracts of mixtures of PS-1-transfectedDAMI cells with β-APP-transfected DAMI cells were subjected to SDS16PAGE and immunoblotting with either anti-PTyr or anti-c-Src antibodies,both antibodies reacted with the same two bands (FIG. 9 a, Panels 1-3).Panel 1 of this figure immunoblotted with anti-PTyr antibodies showstransient increases in tyrosine phosphorylation of the protein bandswith a maximum at 8-10 mins after cell mixing. In Panel 2 the sameextracts immunoblotted with the c-Src antibody show no variation withtime, indicating that the c-Src protein concentration remains unchangedduring the increase in its PTyr levels. An important observation wasthat when ES double-null cells transfected with β-APP (thereforeexpressing only β-APP, but no PS-1 or 2) were mixed with DAMI cellstransfected with PS-1 (therefore expressing only PS-1, but no cellsurface β-APP), the p60 c-Src proteins plus one or two additionalproteins underwent transient increases in PTyr modification at similartimes after mixing (FIG. 9 a, Panel 4) that were seen with theβ-APP-transfected DAMI cells mixed with PS-1-transfected DAMI cells(FIG. 9 a, Panel 1). The PTyr modification results were thereforeassociated with PS-1 and not the cell type that expressed it (see belowfor PS-2).

In order to test further whether c-Src was the member of the tyrosinekinase family that underwent transient tyrosine phosphorylation in theβ-APP:PS-1 interaction, experiments were carried out(autophosphorylation) in which the extracts of the mixed transfectedDAMI cells taken at different times after mixing were treated withanti-c-src antibodies, followed by protein-G sepharose beads. To thebeads was then added γ³² PATP; subsequently the proteins weresolubilized from the beads, and subjected to SDS17 PAGE andautoradiography. The results in FIG. 9 b demonstrate that severaltransient bands appear that are maximally phosphorylated at 8-10 minafter cell mixing, a time course corresponding to the appearance of PTyrin the analogous extracts (FIG. 9 a, Panel 1). Prominent among thesebands is one doublet corresponding to c-Src, confirming that c-Src isactivated transiently in the β-APP:PS-1 intercellular interaction.

The identities of the other phosphorylated bands in FIG. 9 b are notknown. Not all of them are necessarily due to tyrosine phosphorylation;some serine or threonine kinases might have been bound to the c-Src thatwas immuno-reacted with specific anti-pc-Src. Involvement of Lyn but notFyn downstream of β-APP:PS-2 intercellular signaling. When β-APP:PS-2intercellular interactions were carried out with mixtures ofappropriately transfected DAMI cells, an entirely different set ofproteins was PTyr modified than for the β-APP:PS-1 system. Althoughbands were detected by the PTyr antibody that were present at 50-66 kDa,these did not correspond to c-Src on Western blots (FIG. 11 a, Panel 1).Furthermore, when extracts of β-APP:PS-2 mixed cells were firstimmunoprecipitated with c-Src antibodies and the immunoprecipitates werethen autophosphorylated in vitro, no significant increases inphosphorylation at the earlier time points (8-10 mins after mixing) wereseen (FIG. 10 b).

At later time points however, c-Src could apparently be phosphorylatedin these samples indicating that it contributes to increases identifiedin the second later peak of β-APP:PS-2 signaling (FIG. 10 b). Thepossible involvement of other members of the Src kinase family wasinvestigated with molecular weights in the 53-59 kDa range other thanc-Src. Lyn (Mwt 53/56 kDa) and Fyn (Mwt 59 kDa) were two candidate Srckinases that were examined.

Results of Western blot hybridization with anti-Lyn antibodies in FIG.11 a show that Lyn protein concentrations do not change when β-APP:PS-2intercellular interactions are carried out, but afterimmunoprecipitation of the extracts with anti-Lyn antibodies and invitro autophosphorylation of the precipitates, a transientphosphorylation of Lyn with peaks of activity at 8-9 and 17-18 min isobserved, along with other phosphorylated bands (FIG. 11 c). Lynundergoes transient phosphorylation in a pattern that is similar to thePTyr increases seen on Western blots and ELISAs for β-APP:PS-2interaction (FIG. 11 c). Fyn, on the other hand, shows noautophosphorylation in-vitro in the same extracts afterimmunoprecipitation with anti-Fyn antibodies (FIG. 11 d), nor any changein its concentration with time (FIG. 11 b).

Example 3

The following data demonstrates G-protein binding to endogenous PS-1 andPS-2 in extracts of mouse frontal cortex. A 20% homogenate of WT mousefrontal was made in GTPγS solublization/extraction buffer [50 mMHEPES/NaOH pH 7.4, 1 mM EDTA, 1 mM DTT, 1% Triton-X100, 60 mMoctylglycoside, 1× Protease inhibitor mix (1 uMphenylmethylsulfonylflouride, 1 ug/mL antipain, 0.1 ug/mL pepstatin A,0.1 ug/mL leupeptin)]. Measurements of [³⁵S]GTPγS binding were performedon Untreated, PTX treated; and PS-1 and PS-2 immuno-depleted extracts.

For untreated samples, 100 μg of extract was brought up to 100 uL inGTPγS solublization/extraction buffer and mixed with an equal volume ofGTPγS Buffer B (50 mM HEPES/NaOH pH 7.4, 40 μM GDP, 50 mM MgCl₂, 100 mMNaCl) for a total volume of 200 μL. The reaction was started with 50 nM[³⁵S] GTPγS (1250 Ci/mMol; Perkin Elmer) and incubated at RT for 60 min.The reaction was stopped by addition of 20 uL 10× Stopping buffer (100mM Tris-HCl, pH 8.0, 25 mM MgCl₂, 100 mM NaCl, 20 mM GTP).

For PTX treated samples, 100 μg of extract was brought up to 100 μL inGTPγS solublization/extraction buffer and treated with 500 ng/mLactivated PTX in the presence of PTX Buffer (20 mM HEPES pH 8.0, 1 mMEDTA, 2 mM MgCl₂, 1 mM NAD). The sample was incubated for 12 hrs at 30°C. The PTX treated sample was then mixed with an equal volume of GTPγSBuffer B and taken through [³⁵S]GTPγS assay as described above.

Extracts of mouse frontal cortex were immunoprecipitated with a mixtureof polyclonal antibodies to PS-1 and PS-2 (10 uL each) at 4° C.overnight to deplete the samples of PS-1 and PS-2. Protein A agarose (20uL slurry/100 μg protein) was added and samples were and shakenend-over-end at 4° C. for 2 h. The PS-antibody-protein A complex wascentrifuged at high speed for 5 min. Supernatant was recovered in and100 μg aliquots were taken through the [³⁵S] GTPγS assay as described.

Following the GTPγS reaction, 5 uL of either anti-PS-1 or anti-PS-2monoclonal antibodies were added and samples were placed at 4° C.overnight. The antibody-protein complex was bound to 20 μL Protein A/Gagarose (Pharmacia) and samples were placed at 4° C. and shakenend-over-end for 2 hrs. The agarose beads were washed three times withWash Buffer 1 (50 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 1% Triton-X100,1× protease inhibitor mix) and once with each Wash Buffer 2(50 mM HEPES,pH 7.4, 1 mM EDTA, pH 8.0, 0.5% Triton-X100, 1× protease inhibitor mix)and 3(50 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 1× protease inhibitormix). The washed agarose beads were then suspended in 5 mLsscintillation fluid (CytoScint, ICN) and counted on a Beckman Coulter LS6000 SC scintillation counter for 3 min.

FIG. 12 shows the ³⁵S-GTPγS incorporation in extracts of mouse brainthat could be immunoprecipitated with monoclonal antibodies to PS-1,suggesting the co-precipitation of ³⁵S-GTPγS-bound G-protein with theendogenous PS-1. This incorporation was greater than 80% of that foundfor extracts which had been prior depleted of PS-1 and PS-2 by treatmentwith polyclonal antibodies to the two PS proteins, showing specificityof the G-protein:PS-1 binding. Treatment with PTx inhibited the³⁵S-GTPγS incorporation by 60%.

FIG. 13 shows the ³⁵S-GTPγS incorporation in extracts of mouse brainthat could be immunoprecipitated with monoclonal antibodies to PS-2,suggesting the co-precipitation of ³⁵S-GTPγS-bound G-protein with theendogenous PS-2. This incorporation was greater than 85% of that foundfor extracts which had been prior depleted of PS-1 and PS-2 by treatmentwith polyclonal antibodies to the two PS proteins, showing specificityof the G-protein:PS-2 binding. Treatment with PTx inhibited the³⁵S-GTPγS incorporation by 55%. These results demonstrate a specificPTx-sensitive G-protein coupling to endogenous mouse brain PS-1 andPS-2.

Sequences corresponding to the first 16 amino acids of intracellularloop 1 [ic1(1-16)], the remaining 16 amino acids of intracellular loop 1[ic1(17-32)], the entire intracellular loop 2 (ic2), the entireintra-cellular loop 3 (ic3), the first 20 amino acids of the cytoplasmicC-terminal tail (C1-20) and the remaining 19 amino acids of thecytoplasmic C-terminal tail (C21-39) for both PS-1 and PS-2 will besynthesized and HPLC purified to >90% purity. FIG. 14 illustratesintracellular domains of PS. Table 1 shows the sequences that can besynthesized from these domains. In addition, a 20 amino acid controlpeptide synthesized in which the sequences of peptide C1-20 can bescrambled. This peptide is part of the 39 amino-acid sequence identifiedas the binding domain on PS-1 for G_(o).

TABLE 1 PS-1 Cytoplasmic PS-2 Cytoplasmic peptides peptides ic1KSVSFYTRKDGQLIYT KSVRFYTEKNGQLIYT (1-16) (SEQ ID NO: 7) (SEQ ID NO: 12)ic1 PFTEDTETVGQRALHS TFTEDTPSVGQRLLNS (17-32) (SEQ ID NO: 8) (SEQ ID NO:13) ic2 VFKTYNVAVD EVLKTYNVAMD (SEQ ID NO: 9) (SEQ ID NO: 14) ic3MALVFIKYLPE (identical to ic3 of (SEQ ID NO: 10) PS-1) C(1-20)KKALPALPISITFGLVFYFA KKALPALPISITFGLIFYFS (SEQ ID NO: 11) (SEQ ID NO:15) C TDYLVQPFMDQLAFHQFYI TDNLVRPFMDTLASHQLYI (21-39) (SEQ ID NO: 16))(SEQ ID NO: 17)

Example 4

The present studies demonstrate that the GPCR function of PS-1 modulatesthe production of Aβ. A major question in the study of PS-GPCR functionis to determine a specific ligand for PS that can elicit G-proteinactivities from the PS, to which the ligand binds intercellularly. Thepresent studies investigated whether the three-partligand-receptor-G-protein system initiates the production of Aβ. In sucha system activation of PS by ligand (β-APP) binding would lead toG-protein binding to PS in the cytoplasmic domain.

In order to investigate whether G-protein binding to PS-1 or PS-2affects Aβ production from β-APP, cell:cell interaction of β-APP andPS-1 in the presence and absence of Pertussis toxin (PTx) experimentswere performed. PTx is a specific inhibitor of G-protein G_(o)activation. If the GPCR function of PS is involved in the production ofAβ from β-APP:PS intercellular binding, then in its presence, Aβproduction should be inhibited.

β-APP:PS-1 mediated cell-cell interactions were carried out usingmethods described above, with PS-1 transfected primary fibroblasts fromβ-APP−/− mice (cells express PS-1 and do not produce β-APP) interactedwith β-APP-transfected ES (PS−/−) cells (cells produce β-APP but do notexpress PS) in the presence of ³⁵S-methionine. 24 h after co-culture ofthe transfected cells, the samples were harvested in the presence ofprotease inhibitors. Cells were sonicated and 100 μg of whole cellextracts were immunoprecipitated with antibodies to Aβ (6E10) andimmunoprecipitated samples were run on Bicene-Tris gels. Aβ bands werevisualized by autoradiography of dried gels. The same experiment wascarried out in the presence of 500 ng/ml of PTx. Treatment of culturedcells was carried out for 12 h as described below. As a control for PTxtreatment, the cultured cells were incubated with PTx buffer onlycontaining ATP and NAD. Under these conditions activation of Go andlevels of Aβ should be unaffected.

FIG. 15 shows the results of these studies. Lane 1 shows the results ofβ-APP-expressing ES (PS−/−) cells co-cultured with PS-1-expressingFibroblasts (β-APP−/−). Lane 2 shows the results of the components usedin lane 1 in the presence of PTx and PTX buffer (NAD+ATP). Lane 3 showsthe results of the components used in lane 1 in the presence of PTxbuffer only (NAD+ATP), and no PTx. Lane 4 shows the results of tail-lessβ-APP-expressing ES (PS−/−) cells co-cultured with +Tail-lessPS-1-expressing Fibroblasts (β-APP−/−). Lane 5 shows the results of thecomponents used in lane 4 in the presence of PTx. Lane 6 shows theresults of wild type β-APP-expressing ES (PS−/−) cells co-cultured withTail-less PS-1-expressing fibroblasts (β-APP−/−).

The results indicate that PTx toxin inhibits the production of Aβ fromthe intercellular interaction of β-APP and PS-1 (lanes 1 and 2 above).Lane 3 shows that in the presence of PTx buffer only, but in the absenceof PTx, Aβ production is not inhibited. Lanes 4 and 6 show that thecytoplasmic carboxyl terminal domain of PS-1, earlier shown to be thebinding domain of PS-1 for Go, when absent, eliminates Aβ production.

The data provided herein indicate that β-APP is a ligand for PS-1 whichupon binding activates its GPCR activity. The data also indicates thatthe GPCR function of PS-1 is involved in the production of Aβ from β-APPafter its intercellular interaction with PS-1. These results furtherindicate that modulating GPCR activity of PS-1 also modulates theproduction of Aβ. Accordingly, agents that modulate GPCR activity ofPS-1 will modulate the production of Aβ.

For co-culture experiments ES (PS^(−/−)) and β-APP(^(−/−)) cells wereplated at 1×10⁷ cells per 25 cm² flask and transfected with theappropriate cDNAs. 5 hours after transfection, ES(PS-1^(−/−)/PS-2^(−/−)) cells transfected with β-APP were detached bymild trypsinization, washed 2× with met-free culture medium containingheat-inactivated, dialysed FCS (10% v/v) and resuspended in this mediumat 0.33×10⁷ cells/ml. Similarly, primary fibroblasts from β-APP knockoutmice were co-transfected with PS-1 or PS-2 and plated at 1×10⁷ cells.Transfected cells were washed 2× with met free medium and left in 3 mlmet-free medium.

β-APP transfected ES (PS-1^(−/−)/PS-2^(−/−)) cells (1×10⁷ cells/3 mlmet-free medium) were added to the PS-1-transfected β-APP knockoutcells. The cell densities ensured that essentially all the cells were incontact with another. ³⁵S-met (66 μCi/ml; 1175Ci/mmol, NEN) was addedand the cultures incubated for 24 h. In experiments with PTx treatment,500 ng/ml PTx was added to the cultures under the appropriate reactionconditions at this stage and incubated for 24 h. The medium was thenremoved and cells harvested by scraping. A protease inhibitor mix wasadded to the medium before freezing on dry ice. 100 μl extraction buffer(50 mM Tris, pH 8.0/150 mM NaCl/0.5% Nonidet-P40) containing proteaseinhibitors (1 mM 4-(2-aminoethyl) benzene sulfonyl fluoridehydrochloride (AEBSF)/0.1 μg/ml antipain/0.1 μg/ml pepstatin A/0.1 μg/mlleupeptin) was added to the cell pellet and the samples quick-frozen ondry ice.

The PTx protomer (Biomol Research Laboratories) was incubated with 10 mMDTT at 37° C. for 10 min to convert it to its enzymatically active form.5 h after transfecting ES cells with PS-1 or PS-2 and the G-proteincDNAs, 500 ng/ml of activated PTx was added to the cells in culturemedium in the presence of 1 mM NAD, 1 mM ATP, 2 mM MgCl2 and 1 mM EDTA.The cells were incubated at 37° C. in the presence of 5% CO₂ for 18 h.

Whole cell extracts were prepared using cell-pellets sonicated with 3bursts of 20 seconds each on ice. Protein concentration was determinedaccording to the method of Lowry.

Immunoprecipitations were carried out using 100 μg of cell extractsubjected to immunoprecipitation in an end-over-end rotator at 4° C.overnight with 2 μg Aβ specific monoclonal antibodies 6E10 (Senetek),which was raised to residues 1-17 of Aβ (Senetek). 40 μl slurry ofProtein G sepharose (Pharmacia) was then added and allowed to mixend-over-end for 1 h at room temperature. The antigen-antibody-Protein Gsepharose complex was washed once with each of the following: buffer1(10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 0.65M NaCL, 1% NP-40),buffer 2(10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 0.75% NP-40) andbuffer 3(10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 0.1% NP-40). Thewashed complex was boiled for 10 min in bicene-tris sample buffer andsubjected to SDS PAGE on bicene-tris gels.

Bicene-tris gels (15% T/5% C) with 8M urea was cast and run. The gelswere then fixed for 30 min with 5% glutaraldehyde in 0.4M sodiumborate/phosphate buffer and stained for 1 h with 0.1% Coomassie BlueG250 in methanol-acetic acid. After destaining the gels were preparedfor autoradiography.

The destained gels were treated with ethanol (30%) and glycerol (5%) for30 min and impregnated with Amplify (Amersham) for 30 min, dried undervacuum at 80° C. and exposed to X-Omat film at −70° C. for 4-5 days.

Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the teachings of the disclosure or the scope of theinvention as defined by the appended claims. The appendices attachedhereto are provided to further illustrate but not limit the invention.

1. A method of identifying an agent that modulates presenilin-mediatedSrc protein kinase activity, the method comprising: a) contactingpresenilin with β-APP under conditions that would permit binding ofβ-APP to presenilin; b) prior to, simultaneously with, or subsequent toa), contacting presenilin with an agent; c) monitoringpresenilin-mediated Src protein kinase activity; and d) determiningwhether the agent modulates presenilin-mediated Src protein kinaseactivity.
 2. The method of claim 1 wherein the modulating is byinhibition of presenilin-mediated Src protein kinase activity.
 3. Themethod of claim 1 wherein the modulating is by activatingpresenilin-mediated Src protein kinase activity.
 4. The method of claim1 wherein the presenilin is presenilin-1 (PS-1).
 5. The method of claim1 wherein the presenilin is presenilin-2 (PS-2).
 6. The method of claim1, wherein the agent is selected from the group consisting of anaturally occurring or synthetic polypeptide or oligopeptide, apeptidomimetic, a small organic molecule, a polysaccharide, a lipid, afatty acid, a polynucleotide, an RNAi or siRNA, an asRNA, and anoligonucleotide.
 7. The method of claim 1 wherein the contacting is invitro.
 8. The method of claim 1 wherein the contacting is in vivo. 9.The method of claim 6, wherein the synthetic peptide or oligopeptide isa soluble N-terminal domain of PS-1 or -2.
 10. The method of claim 1,further comprising assaying an agent identified as a modulator ofpresenilin-mediated Src protein kinase as an inhibitor of (i) Aβproduction or (ii) the interaction of the N-terminal domain of βAPP withthe N-terminal domain of PS-1 and/or PS-2 comprising contacting a cellsystem comprising a first cell expressing βAPP and a second cellexpressing PS-1 and/or PS-2 with the agent and measuring the presence ofAβ, or the interaction of N-terminal domain of βAPP with the N-terminaldomain of PS-1 and/or PS-2.